Mol bio script 1 (2) (4).pdf

MOLECULAR BIOLOGY
COLLOQUIUM 1, 2 & 3
SCRIPT
Telma Ahmadi 2018, Sem 1

Molecular biology - Colloquium 1 script
Eukaryotes and prokaryotes
Eukaryotes
A eukaryote is an organism are either multicellular or unicellular, in which the genetic
material is organized into a membrane-bound nucleus.
The nucleus structure
The nucleus is protected by the nuclear envelope which is an highly regulated double
layered membrane complex. The membrane consists of phospholid layers that;
Physically separate the nucleus from the cytoplasm
Functionally separate mRNA from the protein synthesis
There is an outer nuclear membrane - shares border with ER, inner nuclear membrane -
encloses the nucleoplasm, and in between is the perinuclear space.
The Nuclear pore complex (NPC) -
NPC is a large protein complex which forms a channel and links the inner and outer
membrane as it is connected by nuclear pores which penetrate the membranes. NPC is
responsible for the protected exchange of components between the nucleus and
cytoplasm.
The central transporter is surronded by 8 nuclear pore groups and connects the
nucleoplasm with the cytoplasm and ensures macromolecular exchange. Small molecules
and ions are transported passively, macromolecules (e.g. proteins, RNA etc.) has active
transport assisted by nuclear transport receptors. Im o in bind im o ing
macromolecules. Exportin binds exporting macromolecules.

LINC - (Linkers of nucleoskeleton and cytoskeleton)
Position the nucleus
Positions the centrosome next to the nucleus
Coordinate nuclear and cytoplasmic acitivies
Involved in mechanical force transmission (from cytosol to nucleus)
Nuclear lamina
The lamina is a structure attached to the inner membrane and the chromatin. There are
two types of laminas:
A or C lamina
Encoded by one gene
B lamina
Encoded by different genes
The nuclear lamina is involved in most nuclear activities:
DNA replication
Transcription
Nuclear and chromatin organization
Cell cycle regulation
Cell development and differentiation
Nuclear migration
Apoptosis (Programmed cell death)
Nucleoplasm
The nucleoplasm is fluid or gel-like substance of the nucleus with suspended chromatin
material, nucleolus, and other particulate elements of the nucleus. The major component
of nucleoplasm are nucleoproteins.
The nucleolus
Inside the nucleus is the nucleolus, it is not membrane-bounded and is the sub-organelle.
The nucleolus contains ribosomal RNA (rRNA) transcription, pre-rRNA processing and
ribosome subunit assembly. Three major components of the nucleolus are recognized: the

fibrillar center (FC), the dense fibrillar component (DFC), and the granular component
(GC).
The fibrillar center -
rRNA genes with RNA synthesis enzymes (transcription)
Dense fibrillar component -
Protein bounded rRNA molecules (pre-rRNA processing);
Granular component -
Pre-ribosomal particles (ribosome subunit assembly).
The storage information in eukaryotes is stored in the nucleus, mitochondria and
chloroplast (plants).
Nuclear DNA (nDNA) is the DNA stored within the membrane-bound nucleus.
It's structure is linear and double-helix. Nuclear DNA is diploid (46
chromosomes).
Mithochondrial DNA (mtDNA) is the DNA stored within the membrane-bound
mitochondria. It's structure is circular and double-helix. Mitochondrial DNA is
haploid (26 chromosomes) only from the mother.
Chloroplast DNA (cpDNA) is the DNA stored within the membrane-bound
chloroplast.
Eukaryotes are divided into three main domains;
Animals (Multicellular)
Animal cells have membrane-bounded nucleus and organelles; such as centrosomes and
mitochondria. Animal cells lack cell wall, due to the lack of the cell wall, the shape and
size of the animal cells are mostly irregular. It also lacks a large vacuole, in contrast,
animal cells have many, smaller vacuoles. Because animals get sugar from the food they
eat, they do not need chloroplasts: just mitochondria.

Plants (Multicellular)
Plant cells have (among other organelles) chloroplasts, mitochondria and large
vacuole. Plan don ge hei ga f om ea ing food, o he need o make ga
from sunlight. This process (photosynthesis) takes place in the chloroplast. Once the
sugar is made, it is then broken down by the mitochondria to make energy for the cell.
Plant cells are surrounded by a cell membrane and a cell wall (surrounds cell
membrane). The cell wall contains celluloses, and the wall gives the cell it's
rectangular shape.
Plant cells contain a large, singular vacuole that is used for storage and maintaining
the shape of the cell.
In plants and animals, there are two major categories of cells: somatic cells and
reproductive cells, known as germ cells or gametes.
Somatic cell take part in the formation of the body, becoming differentiated into the
various tissues, organs, etc. Not involved in reproduction. Somatic cell contain two
copies of genetic information (diploid).
Germ cell a sexual reproductive cell at any stage from the primordial cell to the
mature gamete. Germ cell contain one copy of genetic information (haploid).
Fungus (Unicellular)
Fungus are unicellular, they have mitochondria but lack chloroplasts and centrosomes.
A characteristic that places fungi in a different kingdom from plants is chitin in their cell
walls.

Prokaryotes
Prokaryotes are unicellular organisms that lack membrane-bound nucleus or any other
membrane-bound organelle. Prokaryotes are divided into to domains; bacteria and
archea.
The storage of information in prokaryotes are stored in the nucleoid and plasmids.
In the prokaryotes, all the intracellular water-soluble components
(proteins, DNA and metabolites) are located together in the cytoplasm are enclosed by
the cell membrane, rather than in separate cellular compartments.
The nucleiod
The prokaryote has a nucloid which is an region within the cell of a prokaryote that
contains all or most of the genetic material. The nucleoid is composed of DNA in
association with a number of DNA-binding proteins - and are known as histone-like
proteins - that help it maintain its structure.
The protein HU non-specifically binds to DNA and bends it, with the DNA
wrapping around the protein.
The protein IHF, also facilitates bending of DNA, but it does so by binding to
specific DNA sites.
The protein H-NS binds to DNA and is involved in compacting DNA structure.
The plasmid
The plasmids contain small part of genetic information which contribute to the sensitivity
to various toxic substances like antibiotics.
The DNA in prokaryotes is circular and not attached with histone proteins. The genetic
information has one copy, haploid organism.
Prokaryotic cells & Eukaryotic cells - differences

Prokaryotic Eukaryotic
Cell type Mostly unicellular Unicellular or
multicellular
Cell structure Absence of membrane-
enclosed organelles
Membrane-enclosed
organelles present.
Cell wall Present, chemically
complex
When present
chemically simple
Nucleus Absent.
Contains nucleoid
(nucleus-like)
Present.
Membrane-bound
nucleus and nucleolus
are present.
DNA One or few circular
chromosomes; haploid
genome
Multiple chromosomes;
diploid genome
Storage of information In nucleoid and
plasmids.
In nucleus,
mitochondria and/or
chloroplasts
Ribosomes Small, distributed in
the cytoplasm.
Large, found on
membranes on ER or in
organelles
Viruses, viriods & prions
Viruses, viriods and prions are disease causing agents that must infect host-cell to grow
and reproduce. As they can not grow or self reproduce (only inside the living cells of
other organisms) and lack metabolism they are not considered as living forms. They also
lack cell structure.
Viruses
Viruses are small infectious agents that replicate only inside the living cells of other
organisms. Viruses are cell specific, the host is determined by the capsid protein affinity
to the host-cell receptor. Viruses are able to infect humans, animals, plants and fungus.
While not inside an infected cell or in the process of infecting a cell, viruses exist in the
form of independent particles - also known as virions.
A virus either has a DNA genome (DNA-virus) or RNA genome (RNA-virus). The
genetic material inside the virus are stored inside the nucleic acids, which is protected by
a capsid. Capsid + nucleic acid = nucleocapsid.
Viriods
Viriods are small, circular RNA molecules without a protecting capsid. The hosts of
viriods are plant cells.
Prions
Prions are infectious protein particles without nucleic acid. They infect cells in the
nervous system. The prion protein is a misfolded version of a normal protein in the

nervous system. When the prion infects a nerve it promotes the misfolding of the normal
proteins. Prions cause neurodegenerate disorders in animals and humans.
Nucleic acids
The term nucleic acid is the overall name for DNA and RNA. They are composed
of nucleotides, which are the monomers made of three components:
5-carbon sugar
Phosphate group
Nitrogenous base. Pyrimidines: Uracil, Thymine, Cytosine. Purines: Adenine,
Guanin
If the sugar is a compound ribose, the polymer is RNA (ribonucleic acid); if the sugar is
derived from ribose as deoxyribose, the polymer is DNA (deoxyribonucleic acid).
DNA RNA
BASE A, G, C, T A, G, C, U
PENTOSE Deoxyribose Ribose
PHOSPHATE Phosphate group Phosphate group
STRUCTURE Double-stranded Single-stranded
DNA -
Deoxyribonucleic acid is composed of two chains
(made of nucleotides) that coil around each other to
form a double-helix.
The nucleotide bases (AGCT) of DNA represent the
stairsteps of the staircase. The bases are held
together by hydrogen bonds (2 hydrogen bonds
between Adenine and Thymine, 3 hydrogen bonds
between Guanine and Cytosine). The bases are
hydrophopic (lack affinity for water) and therefore
avoid contact with cell fluids (cytoplasm and
cytosol). The bases are stacked to prevent contact
with cell fluids and this structure stabilizes the
double-helix.
The deoxyribose (5-carbon sugar) and phosphate
molecules form the sides of the staircase and are
hydrophilic (attracted to water) and therefore are on
the outside in contact with the cell fluids. To further
avoid contact between the bases and cell fluids the

DNA is twisted to reduce space between bases and staircase.
The two bonds that attach a basepair to its deoxyribose sugar ring are not directly
opposite therefore the sugar-phosphate backbone is not equally spaced. The double-helix
is antiparallel. This results in major and minor grooves of DNA. Both grooves provide
opportunities for base-specific interactions. E.g. TATA-box binds to specific A och T
rich regions, which results in the unwinding and bending of DNA.
Circular DNA -
There is circular DNA which are found in prokaryotes and in some viruses. The mtDNA
in mitochondria (eukaryotic cells) is also circular.
RNA -
The chemical structure of DNA is very similar to the structure of DNA, but differs in
three primary ways:
RNA is a single-stranded molecule. But it can by complementary base-pairing as
in tRNA, form double-helix strands.
The pentose group compound is ribose.
The bases are: AGCU
The structure of RNA depends on it's functions. Generally:
Primary structure: Single stranded.
Secondary structure: Includes single stranded, double stranded and loop stranded.
Tertiary structure: Able to form (depends on function)
Biological functions of DNA and RNA -
DNA is vital for all living beings even plants. It is important for inheritance, coding for
proteins and the genetic instruction guide for life and its processes. The flow of genetic
info ma ion in a cell i f om DNA h o gh RNA o o ein : DNA make RNA make
protein . Proteins are the workhorses of the cell; they play leading roles in the cell as
enzymes, as structural components, and in cell signaling, to name just a few. DNA is
con ide ed he bl e in of he cell; i ca ie all of he gene ic info ma ion e ired for
the cell to grow, to take in nutrients, and to propagate. RNA in this role i he DNA
ho oco of he cell. When he cell need o od ce a ce ain o ein, i ac i a e he
o ein gene the portion of DNA that codes for that protein and produces multiple
copies of that piece of DNA in the form of mRNA. The multiple copies of mRNA are
then used to translate he gene ic code in o o ein h o gh he ac ion of he cell o ein
manufacturing machinery, the ribosome.
In addition to mRNA, rRNA and tRNA, which play central roles within cells, there are a
number of regulatory, non-coding RNAs (ncRNAs). Of varying lengths, ncRNAs have no
long open reading frame. While not encoding proteins, they may act as riboregulators,
and their main function is posttranscriptional regulation of gene expression.
DNA replication

1) The process is catalyzed by specific enzyme helicase. Helicase uncoils the DNA
double-helix through cleavage of the hydrogen bonds between the nucleotides. The Y-
fork is formed in this stage. Topoisomerase prevent DNA from supercoiling. Single
strand binding proteins prevent reformation of DNA double helix.
2) The DNA replication is then started by the enzyme primase. Primase synthesizes
RNA primers that are attached to the single stranded DNA. The primer always binds as
the starting point of replication.
3) The DNA polymerase is also responsible for creating the new strand by a process
known as elongation. Each strand of the original DNA molecule serves as a template for
the production of its counterpart. The new strand is synthesized by the attachment of free
dNTP at the site of the primer by the DNA polymerase.
4) The strands of the double helix are anti-parallel with one being 5' to 3', and the
opposite strand 3' to 5', whereas a new strand is always n he i ed in he 5 o 3
direction (You can only extend DNA to 5' prime in 3' direction) -> semi-conservative
replication. The leading strand is the strand of new DNA which is being synthesized in
the same direction as the growing replication fork. This sort of DNA replication is
continuous. DNA ol me a e e ilon ( ) n he i e ne and f om leading and. The
lagging strand however is the strand of new DNA whose direction of synthesis is
opposite to the direction of the growing replication fork. The lagging strand whose
direction of synthesis is opposite begins replication by with multiple RNA primers added
by the DNA primase, which allows the DNA ol me a e del a ( ) to add dNTP between
the primers in the 3' to 5' prime direction. This process of replication is discontinous as
the newly created fragments, called okazaki fragments, are disjointed.

5) Once both the continous and discontinous strands are formed, we need to remove the
primers which helped to initiate the polymerase process. RNase H recognise and degrade
RNA ime af e ne DNA and i n he i ed. DNA ol me a e be a ( ) fill in
empty space with appropriate bases. DNA-ligase joins the okazaki fragments creating a
unite single strand.
6) After replication of double stranded DNA, problem arise on lagging strand - when the
replication fork reaches the end of the chromosome there is a short stretch of DNA that
does not get covered by an Okazaki fragment because there is no free 3` OH group to
attach the phosphate group of an incoming nucleotide. Part of the DNA at the end of a
eukaryotic chromosome goes uncopied in each round of replication, leaving a single-
stranded overhang.
To prevent the loss of genes as chromosome ends wear down, the tips of eukaryotic
chromosomes have specialized DNA ca called telomeres. Telomeres consist of
hundreds or thousands of repeats of the same short DNA sequence, which varies between
organisms but is 5'-TTAGGG-3' in humans and other mammals.
Some cells have the ability to reverse telomere shortening by the enzyme telomerase.
This acts as a template for single stranded telomere cap synthesis.
Replication in eukaryotes VS. prokaryotes
The process of DNA replication in prokaryotes and eukaryotes share some similarities
and differences
Prokaryotes Eukaryotes
Place Cytoplasm Nucleus
Place of origin One Multiple

Replication fork Bidirectional In parallel in many
origins of replication
Okazaki fragments Long Short
Speed of synthesis Fast Slow
Eukaryotic chromosomes
Cell cycle
INTERPHASE
The interphase is the longest phase.
In this phase the chromosomes are in
their chromatin form (unwound).
During interphase, the chromatin is
structurally loose to allow access
to RNA and DNA polymerases that
transcribe and replicate the DNA.
The local structure of chromatin
during interphase depends on the
genes present on the DNA. That
DNA which codes genes that are
actively transcribed ("turned on") is
more loosely packaged and
associated with RNA polymerases
(referred to as euchromatin) while that DNA which codes inactive genes ("turned
off") is more condensed and associated with structural proteins
(heterochromatin). The local chromatin structure, in particular chemical
modifications of histone proteins is done by methylation and acetylation. In the
(S) phase the DNA replicates two chromosome copies are made called sister
chromatids which are connected by the centromere. Cohesin attaches to the
chromosome during replication and ensure sister chromatid contact during cell
division.
Mitosis
In this phase the DNA goes into more condensed form. At the time of cell
division the chromatin is in it's most condensed phase, this structure is called the
metaphase chromosome. The metaphase chromosome is formed by the help of the
protein complex: condensis. Condensis has a impact on chromosome
condensation and stabilization during the cell divison. Nuclear membrane starts to
fade, the centrosomes go to opposite directions of the cell. Then the chromosomes
start lining up in the middle of the cell, the membrane is completely gone, the
centrosomes have extended microtubules that are connected to centromeres. The
centromere forms site for kinetochore attachment, the kinetochore are large,
multiprotein complex that is needed to link the sister chromatids to the mitotic
spindle during chromosome segregation. The metaphase chromosome can be
observed during cell division.

The cell splits into two -> cytokinesis, nuclear membrane starts to form, DNA
back to chromatin form
Chromosome structure
Centromere; hold the sister chromatins
together and point of attachment of spindle
microtubules (kinetochore; protein complex to
which the spindle microtubules attach)
Telomeres; Natural ends of linear
chromosome. Telomeres consist of repeated
nucleotide sequence which are species
specific. It's function is to stabilize the
chromosome ends.
Satellites; round bodies separated from the
rest of the chromosome by secondary
constriction; tandemly repeated DNA
sequences presented as long uninterrupted
arrays in genetically silent heterochromatic
regions
Secondary constriction; a subsidiary
narrowing of the chromosome associated in
some cases with satellites.
Nucleolus organizing regions (NOR)
chromosomal region which forms nucleolus after cell division.
Each chromosome has two arms, labeled p (short) or q (longer). They can either be
connected in: metacentric, submetacentric or telocentric manner.
Metacentric -
These are x-shaped chromosomes with the centromere in the middle so that the arms of
the chromosomes are almost equal.
Submetacentric -
If the arms are unequal, the chromosomes are said to be submetacentric.
Acrocentric -
If the ''p'' arm is so short that it is hard to observe, but still present, then the chromosome
is acrocentric.
Telocentric -
Centromere is located at the terminal end of the chromosome. Telomeres may extend
from both ends of the chromosome.
The number, sizes, and shapes of the metaphase chromosomes constitute the karyotype,
which is distinctive for each species. Normal human karyotype:
Somatic cells have diploid set of chromosomes (2n) 46 chromosomes;
Germ cells have haploid set of chromosomes (1n) 23 chromosomes;

The chromosome which determine the sex and sex-linked characteristics of an
organism is called sex chromosome. Humans have two sex chromosomes X
chromosome and Y chromosome. Each individual cells contain two sex
chromosomes in diploidic cell. Female XX and male XY.
Any chromosomes that is not a sex chromosome autosome. Same for all
individuals
Female Male
Somatic cells 46, xx 46, xy
Germ cells 23, x 23, x or 23, y
Barr body One x chromosome is
inactivated (Barr body) in all
somatic cells
X chromosome is activated,
no barr body
Barr body - inactive x chromosomes. There is always one active x chromosome - rest are
barr bodies.
Nucleoproteins
The protoplasm of the nucleus of a cell is called nucleoplasm (karyoplasm). The fluid
or gel-like substance of the nucleus contains of suspended chromatin material,
nucleolus, and other particulate elements of the nucleus. The major component of the
nucleoplasm are nucleoproteins;
Histones -
Histones are a family of basic proteins that associate with DNA in the nucleus and help
condense it into chromatin, they are alkaline (basic pH) proteins, and their positive
charges allow them to associate with DNA (negatively charged). DNA and histones are
packed together to be nucleosome, nucleosome form a pack which are called chromatin,
two chromatin form a chromosome.
The five major families of histones are: H1, H2A, H2B, H3, H4
Non-histones - Small, acidic proteins, such as enzymes. The total precantage of non-
histones in the nucleoplasm is low.

Chromosome -
Chromatin is complex of DNA and nucleoproteins. It has long, stretched and coiled linear
structure. This structure is also called interphase chromosome.
Metaphase chromosome is formed by the help of protein complex condensins.
Condensins have impact on chromosome condensation and stabilization during cell
division. Cohesin attaches to the chromosome during replication and ensure sister
chromatid contact during cell division.
Majority of DNA in eukaryotes is packed into nucleosomes. The nucleosome is
composed of eight histone molecules (H2A, H2B, H3 and H4). Linker DNA tightly
binds with histone H1.
During interphase (DNA in chromatin form, it is not twined) two types of chromatin can
be distinguished; Eurochromatin and heterochromatin;
Eurochromatin -
Lightly packed form of chromatin that is enriched in genes; high level of gene
expression. 92% of the human genome is eurochromatin.
Heterochromatin -
Tightly packed form of chromatin that has little or no gene expression. It is also
associated with telomere and centromere regions of chromosomes. DNA of
heterochromatin is methylated (gene silencing). Heterochromatin consist of constitutive
heterochromatin and facultative heterochromatin.
a) Constitutive heterochromatin
Contains highly repetitive sequences of genetically inactive DNA. Constitutive
heterochromatin is composed mainly of high copy number tandem repeats known as
satellite repeats, minisatellite and microsatellite repeats, and transposon repeats. Forms
structural functions such as centromeres or telomeres
b) Facultative heterochromatin
The facultative heterochromatin is reversible, and can become transcriptional active but
also be heterochromatin in certain cells/tissues. An example of facultative
heterochromatin is X chromosome inactivation in female mammals: one X
chromosome is packaged as facultative heterochromatin and silenced, while the other X

chromosome is packaged as euchromatin and expressed. It is characterized by the
presence of LINE-type repeated sequences.
Functions of heterochromatin:
Centromere function
Organisation of nuclear domains
Gene repression (epigenetic regulation)
Prokaryotic chromosome
The DNA in the prokaryotic cell is stored in the nucleoid and the plasmids.
The DNA in the prokaryotic chromosome is circular, and not attached with histone
proteins. The genetic information has one copy haploid organism (set of 26
chromosomes).
Most prokaryotes do not have histones (with the exception of those species in
the domain Archaea). Thus, one way prokaryotes compress their DNA into smaller
spaces is through supercoiling. When this type of twisting happens to a bacterial genome,
it is known as supercoiling. Genomes are negatively supercoiled, meaning that the DNA
is twisted in the opposite direction of the double helix.
Two DNA topoisomerases control the level of negative supercoiling in prokaryotic cells:
DNA gyrase introduces supercoils, and DNA topoisomerase I prevents supercoiling from
reaching unacceptably high levels.
Mitochondrial chromosome/organelle
Although most DNA is packaged in chromosomes within the nucleus, mitochondria also
have a small amount of their own DNA. This genetic material is known as mitochondrial
DNA or mtDNA.
mtDNA is circular, negatively supercoiled, similar as bacterial chromosome and lies
in the matrix.
A mitochondria contains outer and inner membranes composed of phospholid bilayers
and proteins. The two membranes have different properties.
1. The outer mitochondrial membrane
This part encloses the entire organelle. The outer membrane is composed of equal
amounts of phospholipids and proteins. It is permeable to nutrient molecules, ions,
ATP and ADP molecules. This transport is ensured by porines.
2. The intermembrane space
The space between outer membrane and inner membrane. It is similar to cytosol
3. Inner membrane
The inner membrane is folded into cristae to increase the surface areas inside the
organelle. Contribute to various chemical reactions (e.g. ATP production). Is strictly
permeable - only to oxygen, ATP and regulated transfer of metabolites across the
membrane.
4. Matrix

The matrix is the spaced enclosed by the inner membrane. The matrix is important for
the synthesis of ATP molecules, mitochondrial ribosomes, tRNAs and mitochondrial
DNA (mtDNA).
Functions of mitochondria:
Self-replication;
Cytoplasmic inheritance - transmission to the daughter cells via cytoplasm;
Aerobic ATP production
Ion homeostasis and storage of calcium ions
Biogenesis of steroids - important roles in biosynthesis of steroid sex hormones;
Apoptosis - contain several pro-apoptotic molecules that activate cytosolic
proteins to induce apoptosis.
Genome
A genome is an organism's complete set of DNA, including all of its genes.
Each genome contains all of the information needed to build and maintain that organism.
The genome size is related to the complexity of organism (with some exceptions).
The total genetic content is contained differently in different organisms:
The total genetic content in eukaryotes is contained in a haploid set of
chromosomes
The total genetic content in prokaryotes is contained in a single chromosome in
The total genetic content in viruses is contained in the DNA or RNA.
Codons
Cells decode mRNAs by reading their nucleotides in groups of three, called codons.
There are 4 nitrogenous bases - 3 set is a codon - 4x4x4=64 permutation that correspond
to the 20 amino acids used for protein synthesis and as the signals for starting and
stopping protein synthesis. Here are some features of codons:
Most codons specify an amino acid
One "start" codon, AUG, marks the beginning of a protein and also encodes the
amino acid methionine.
Three "stop" codons mark the end of a protein.

In RNA; UAG, UAA, UGA. In DNA:TAG,TAA,TGA
Codons in an mRNA are read during translation, beginning with a start codon and
continuing until a stop codon is reached. mRNA codons are read from 5' to 3' , and they
specify the order of amino acids in a protein from N-terminus (methionine) to C-
terminus.
Genetic code
The full set of relationships between codons and amino acids (or stop signals) is called
the genetic code. The genetic code is the set of DNA or RNA sequences that determine
the amino acid sequence used in the synthesis of an organisms proteins.
Human genome demonstrate complex structure with low density of genes.
Eukaryotic genome
A gene is chromosomal DNA sequence required for synthesis of a functional protein or
RNA molecule. In addition to the coding regions (exons), a gene includes transcription-
control regions and introns. Although the majority of genes encode proteins, some encode
tRNAs, rRNAs, and other types of RNA.
Promoter region
At the 5` end of the gene lies a promoter region, which includes sequences responsible
for the proper initiation of transcription. These transcription factors have
specific activator or repressor sequences of corresponding nucleotides that attach to
specific promoters and regulate gene expression.
TATA-box
Located upstream (usually 25-30 bp upstream) of transcription start is the TATA-box.
The TATA-box is considered a non-coding DNA sequence that contains repeated T
and A base pairs.

Function; Important for determining the position of start of transcription with help of
the TATA-box binding protein.
CPG-islands
Located upstream (usually 100 bp upstream) of transcription start is the CPG-islands.
The CPG-islands is a DNA sequence rich of C and G. Usually found in housekeeping
genes, tissue-specific genes and developmental regulator genes.
Function; Binds some transcription factors and are targeted for DNA methylation
(DNA methylation is a process by which methyl groups are added to the DNA
molecule. Methylation can change the activity of a DNA segment without changing
the sequence. When located in a gene promoter, DNA methylation typically acts to
repress gene transcription), which lead to the repression of gene expression.
LCR (locus control region)
Controls expression: The h man -globin locus located on a short region
of chromosome 11, responsible for the creation of the beta parts of the oxygen
transport protein Hemoglobin. Expression of all of these genes is controlled by
single locus control region (LCR), and the genes are differentially expressed
throughout development.
The alpha-globin locus located on chromosome 16.
Function: Ability to enhance the expression of linked genes (When two genes are
located on the same chromosome they are called linked genes because they tend to be
inherited together. )
5`UTR (untranslated region)
Initiator element (initiator sequence, Inr)
Regulatory sequences to promote ribosome binding with mRNA
Exon
Coding sequence of a eukaryotic gene's DNA that uninterrupted transcribes into protein
structures
Intron
Noncoding DNA sequence of a eukaryotic gene's that is not translated into a protein.
ORF - open reading frame
The sequence from the start codon to the stop codon is called ORF. Typically only one
ORF is used in translating a gene and this is often the longest ORF.
To find the ORF;
1) Write complementary strand of DNA
2) Look for the longest possible ORF in both strands
3' UTR (untranslated region)
The section that follows termination codon (stop)
Regulating sequences to promote rapid degradation of mRNA
Regulate levels of translation
Polyadenylation [poly(A)] signals (PAS) -

Se e a 3 -end cleavage and polyadenylation of pre-mRNA
Promote downstream transcriptional termination.
Transcriptional enhancers and repressors
In some eukaryotic genes there are regions that increase or enhance transcription.
Enhancers'-
The regions called enchansers are not necessarily close to the genes they enchanse. They
can be located both upstream, within the coding region, downstream or thousands of
nucleotides away.
Enhancer regions are binding sequences for transcription factors. When a DNA-bending
protein binds to the enhancer the shape of the DNA changes which allows interactions
between activators (bound to the enchancers) and the transcription factors (bound to the
promoter and the RNA polymerase) to occur.
Repressors/silencers
Transcriptonal repressors can bind to a promoter or enhancer region and block
transcription. Repressors respond the external stimuli to prevent the binding of activating
transcription factors.
Organisation of human genome
Human genome consist of nuclear genome and mitochondrial genome.
Human nuclear genome: 2 sections
Intergenic region - 75% of human genome.
Stretch of DNA sequences between genes (different to introns that are within genes),
subset (delmängd) of non-coding DNA. Tandemly repeated genes encoding rRNAs,
tRNAs, snRNAs, and histones - multiple tandemly repeated genes encode identical
or nearly identical proteins or functional RNAs.
- Repetitious DNA
Repetitious sequences are patterns of nucleic acids tat occur in multiple copies
throughout the genome
a) (Simple repetitive)
Tandem repeats - TAG's - Gene clusters created by tandem duplications. A process
in which one gene is duplicated and the copy is found adjacent (right next to) the
original -> Tandem arrangement. There are three categories:
1. Satellite
Satellite DNA consists of very large arrays of tandem repeated, non coding DNA. It is
the main component of functional centromeres and form the main structure of
heterochromatin.
2. Mini-satellite DNA
Mini-satellite DNA is the section of DNA that consists of short series of repeated
bases (6-64 bp). Generally GC rich. Mini-satellites constitute the chromosomal
telomeres and subtelomeres.
3. Micro-satellite DNA
The shortest section of tandem repeated bases (1-4 bp long). They are dispersed
throughout all chromosomes.

- Transposone
Transoposones are a group of transoposable elements that are mobile DNA sequences
that can migrate to different regions of the genome. It's function is important for genome
plasticity (quality of being easily shaped or moulded) by promoting interchromosomal
crossing-over or intrachromosomal recombination, leading to deletions
(removal)/duplications or inversions (changed to opposite of what it was). Insertion into
genes can disrupt the gene function and cause an inherited diseases.
This function is either directed by a copy-and-paste mechanism or through an RNA
intermediate. There are four different transpones, whereas 3 are retro-trasposones (Copy-
and-paste mechanism as it converts an RNA transcript of itself into cDNA (copy DNA)
that then integrates into DNA at different location)
a) SINE
Short interspersed nuclear elements (100-400 bp in length). Retrotransposon. Do not code
any proteins.
b) LINE
Long interspersed nuclear elements. There are three LINE families. Retrotransposon.
Encodes two enzymes.
c) LTR
Long terminal repeats. Retrotransposon.
d) DNA transposone
A group of transposable elements that can movie into the DNA via copy-and-paste
mechanism but during cell division. This segment on the chromosome divides and
''jumps'' to any random position in the chromosome during division.
-Unique DNA
A unique DNA is a stretch of DNA present in only a single copy in a cell
Genes and related sequences: 25% of human genome
Coding and regulatory regions
- Protein coding genes:
1) solitary genes
Solitary genes only have one copy in the haploid genome
2) duplicated genes
Duplicated genes are genes with close but not identical sequences, located within a
specified distance of one another. A set of duplicated genes is called a gene family.
Introns, promoters, pseudogenes
- Pseudogenes
A pseudogene is a non-functional gene copy, a defective that contains at least multiple
exons of a functional gene.
Changes in genome may lead to the human pathology
Fragile X syndrome -

Nearly all cases of fragile X syndrome are caused by a mutation in which a DNA
segment, known as the CGG triplet repeat, is expanded within the FMR1 gene.
Normally, this DNA segment is repeated from 5 to about 40 times. In people
with fragile X syndrome, however, the CGG segment is repeated more than 200
times. The abnormally expanded CGG segment turns off (silences) the FMR1 gene,
which prevents the gene from producing FMRP. Loss or a shortage (deficiency) of
this protein disrupts nervous system functions and leads to the signs and symptoms
of fragile X syndrome.
Mitochondrial DNA genome
The human mitochondrial genome consists of a circular double-stranded DNA, and
contains 37 genes.
Mitochondrial DNA has two DNA strands designated as the heavy strand and the light
strand. The heavy strand is rich in (G)uanine and encodes 28 genes. The light strand is
rich in Cytosine and encodes only 9 genes.
Prokaryotic genome
The prokaryotic genome is circular, double-stranded DNA. Most prokaryotes have very
little repetitive DNA in their genomes. Also few transposable sequences.
The prokaryotic gene structure is simpler compared to eukaryoes. It does not contain
introns and the promoter region consist of two regulatory sequences (-10bp Pribnow box;
-35 bp element):
In prokaryotes the genes are encoded together within the genome in a block called an
operon, they are grouped based on similar functions into functional units - which are
regulated together. The operons are transcribed together under the control of a single
promoter (Regulatory DNA sequence). The promoter has simultaneous control over the
regions of the transcription of these genes because they are all needed in the same time.
A DNA sequence called the operator is encoded between the promoter region and the
first codon gene, this operator contains the DNA code to which the repressor protein

can bind (and stop the transcribing). There are also proteins that bind to the operator that
act as an positive regulator to turn genes on and activate them.
The lac operon is an example on an inducible operon - it is usually off but when a
molecule called an inducer is present the operon turns on. It's function is to produce
enzymes which break down lactose (milk sugar). When lactose is present, they turn on
genes and produce enzymes. It has two components – repressor gene (lacI) and 3
functional genes (lacZ, lacY, lacA).
Ex. 1) No lactose
When there is no lactose present, the functional genes are not transcribed into enzymes
that can metabolize and absorb the lactose. This is regulated through the lac repressor
(Lacl) protein that binds to the operator.
Ex 2) Lactose
When lactose is present, the Allo lactose is also present, the Allo lactose can act as an
inducer of transcription through binding to the lactose repressor. When Allo lactose binds
to the lactose repressor it can no longer bind to the operator. And so the RNA polymerase
can transcribe the genes and lactose can be metabolized.
Gene functions
A constitutive gene is a gene that is transcribed continually as opposed to a facultative
gene, which is only transcribed when needed.
A housekeeping gene is typically a constitutive gene. The level of transcription is
relatively constant, not influenced by environmental factors. The housekeeping gene's
products are typically needed for maintenance of the cell; gene expression, metabolism,
structural, surface, signalling etc. Structure: Promoter region is relatively simple, do not
contain TATA box. Shorter introns and exons
Facultative genes genes with inducible expression, based on stage of development, cell
cycle, as a response to environmental factors etc. Often tissue-specific genes. Structure:
Promoter region more complicated, with multi-step regulation, contain CpG island.
Gene expression
Gene expression is the translation of information encoded in a gene into protein or RNA
structures. It involves two steps:
1. Transcription -
The process of making copy of genetic information stored in a DNA strand into a
complementary strand of mRNA with the aid of DNA polymerase.
2. Translation -
A step in protein synthesis whereas the coded info carried by the mRNA is decoded to
produce the specific sequence of amino acids in a polypeptide-chain.
Gene expression in eukaryotes (Transcription)
The transcription process is divided into three phases; initiation, elongation and
termination.
1. Transcription
INITIATION PHASE

During the initiation phase the RNA polymerase II bind to the promoter sequence (5'UTR
region) in the double-stranded DNA. The polymerase then melts the DNA, which forms
the transcription bubble. The DNA has now turned from a closed complex to a open
complex. In the next step the polymerase begins polymerization of ribonucleotides
(rNTPs) at the start site, which is located within the promoter 5`UTR region.
Transcription initiation is considered complete when the first two ribonucleotides (rNTP)
are linked by a phosphodiester bond (the linkage is catalyzed by the RNA polymerase)
ELONGATION PHASE
In the elongation phase RNA polymerase moves along the template DNA one base at a
time. Opening the double stranded DNA in front of it's direction and hybridizing (form
base pairs) the strands behind it.
The direction of the synthesis of the RNA strand is 5'3 while the direction of the strand
being transcribed is 3'5. Approximately eight nucleotides at the 3` end of the growing
RNA strand remain base-paired to the template DNA strand in the transcription bubble.
TERMINATION PHASE
During termination the completed RNA, pre-mRNA, is released from the RNA
polymerase and the polymerase dissociates from the template DNA. AATAAA sequence
is a signal for the bound RNA polymerase to terminate transcription.
RNA-PROCESSING
The pre-mRNA must undergo several modifications termed as RNA-processing to form a
functional matured mRNA.
At the 5` end of a growing RNA chain appearing from the surface of RNA polymerase II,
it is immediately acted on by several enzymes that together synthesize the 5` cap in a
process called capping. A nucleotide called 7- methylguanylate is added to the 5'prime
end of the pre-mRNA.
(The cap: protects an mRNA from enzymatic degradation, assists in export to cytoplasm,
has role in initiation of translation in cytoplasm)
The 3'end of the pre-mRNA is also processed. The process involves cleavage by an
enzyme called endonuclase to provide a free 3'OH-group to which adenosines are added.
The adenosines are added by an enzyme called poly(A) polymerase. The
poly(A)polymerase tail consists of 150-200 bp of adenosines.
The final step in the processing of mRNA molecules is RNA splicing.
The internal cleavage of a transcript to remove the introns and ligation of the coding
exons is a process called splicing. Splicing is ensured by a protein complex called
spliceosome.
The spliceosome recognizes where the exons finish and introns start by the marking of
specific nucleotide sequences. The end of the first exone - AG The s ar of he firs
intron - G End of firs in ron - AG S ar of ne e one - G (If this is cleaved right
we will still have AG-G sequence on ligated exons)

Alternative splicing; Alternative splicing is a process by which exons within pre-mRNA
transcript are differently joined or skipped. This results in multiple protein isoforms
being encoded by a single gene. mo e o ein f om le gene .
Alternative splicing generates a great amount of proteomic diversity in humans and
significantly affects various functions in cellular processes; tissue specificity,
development state and disease conditions.
Rule; the first and last exon can not be skipped.
Because the first exon contains the start codon, meanwhile the last one contains the stop
codon.
Matured mRNA is now exported through the nuclear pore complex to the cytoplasm.
2. Translation
INITATION
The translation begins with the binding of the small ribosomal subunit to a specific
sequence on the mRNA chain. The small subunit binds via complementary base pairing
between one of its internal subunits and the ribosome binding site.
A specific tRNA-Met (carrying the anticodon methionine - UAC) molecule recognizes
the start/initiator codon (AUG) on the mRNA and binds to it.
Next the large subunit binds, and the tRNA-met is placed in the P-site (protein/peptidyl)
The initiation complex is now formed.
ELONGATION
Our first, methionine-carrying tRNA starts out in the middle slot of the ribosome, called
the P site. Next to it, a fresh codon is exposed in another slot, called the A site. The A site
will be the "landing site" for the next tRNA, one whose anticodon is a perfect
(complementary) match for the exposed codon. The rRNA (large ribosomal subunit)
helps the formation of the peptide bond that connects one amino acid to another. Once the
peptide bond is formed, the mRNA is pulled onward through the ribosome by exactly one
codon. This shift allows the first, empty tRNA to drift out via the E ("exit") site. It also
exposes a new codon in the A site, so the whole cycle can repeat.
TERMINATION
Translation ends in a process called termination. Termination happens when a stop codon
in the mRNA (UAA, UAG, or UGA) enters the A site. Stop codons are recognized by
two proteins called release factors, which also fit into the P site. Release factors mess
with the enzyme that normally forms peptide bonds: they make it add a water molecule to
the last amino acid of the chain. This reaction separates the chain from the tRNA, and the
newly made protein is released.
The sequence of nucleotides in DNA has now been converted to the sequence of amino
acids in a polypeptide chain.
Gene expression is eukaryotes VS. prokaryotes
The main difference between gene expression in eukaryotes and prokaryotes are due to
the difference in cell structure.

Prokaryote Eukaryote
Both transcription and translation
take place in the cytoplasm
Transcription takes place in the
nucleus, translation occurs in the
cytoplasm
Both transcription and translation
are continuous and occur
simultaneously
Transcription and translation are
separate processes that occur
divided in time and space
One type of RNA polymerase
enzyme synthesize all types of RNA
in the cell. Less complex structure;
3 RNA polymerases with complex
structure;
The pre-mRNA has few extra
nucleotides;
Complex pre-mRNA processing
with large number of extra
nucleotides;
One or more genes are transcripted
at a time
One gene is transcripted at a time
Requires 3 initiation factors Requires 9 initiation factors;
Requires 3 release factors; Requires 2 release factors;
Little processing of mRNA Processing of 5` cap and 3` poly(A)
tail.
Proteins
Protein function is derived from three-dimensional structure, and three-dimensional
structure is specified by amino acid sequence.
1. Primary structure - the amino acids linked by peptide bonds into linear chain, based on
mRNA sequence.
2. Secondary structure - The next level of protein structure, secondary structure, refers
to local folded structures that form within a polypeptide due to interactions between
atoms of the backbone. The mo common e of econda c e a e he heli
and he pleated sheet. Both structures are held in shape by hydrogen bonds

3. Tertiary structure - the overall conformation of a polypeptide chain: the three-
dimensional arrangement of all its amino acid residues
The proper folding of proteins within cells is mediated by proteins called chaperones.
Chaperone binds to the growing chain stabilizing it in an unfolded configuration until
synthesis is completed. The completed protein is then released from the ribosome and is
able to fold into its correct threedimensional conformation.
To form the structure of proteins there are enzymes involved. Two types of enzymes
isomerases:
catalyse protein folding by breaking and re-forming covalent bonds
Form disulphide bonds between cysteine residues
Catalyse the isomerization of peptide bonds between proline residues
Important in stabilizing the folded structures of many proteins
Proteins are modified by the addition of a sugar - a process called glycosylation.
Glycosylation is initiated in the endoplasmic reticulum before translation is complete.
Some proteins (in eukaryotes) are modified by the attachment of lipids to the polypeptide
chain.
An important step in the maturation of many proteins is cleavage of the polypeptide
chain, this process is called proteolysis. Proteolysis is the hydrolysis (separation of a
larger molecule into component parts) of the peptide bonds that hold proteins together,
resulting in the breakdown of proteins into their key components peptides and
amino acids.
Proteolysis in organisms serves many purposes; Proteolytic modifications play role in the
translocation of many proteins across membranes by cleavage of aminoterminal

sequence. It also has a function in regulation of cellular processes by reducing the
concentration of a protein.
Some proteins are only required for a specific, time-limited purpose and must be
degraded once their purpose has been served. But errors also frequently occur during
production and folding. These defective proteins are not functional and can even harm the
organism. Therefore they, too, must be degraded. The cells therefore have a sophisticated
system to dispose of defective, superfluous proteins. In the ER there is a special process
for protein degradation, known as ER-associated degradation (ERAD). This system
contains a number of enzymes that cooperate to ensure that a defective protein is marked
with a molecular tag. A protein tagged with such a molecular chain is transported to the
proteasome, the protein-cleaving machinery of the cell, where it is separated into its
components through hydrolysis. Defective proteins that escape this system trigger serious
diseases such as Alzheimer's, Parkinson's, Huntington's disease, cystic fibrosis or
diabetes
Human protein-folding diseases
1-antitrypsin deficiency
Alpha-1 antitrypsin is a protein that protects the body from a powerful enzyme called
neutrophil elastase. Neutrophil elastase is released from white blood cells to fight
infection, but it can attack normal tissues (especially the lungs) if not tightly
controlled by alpha-1 antitrypsin.
Changed forms of this protein fail to complete proper folding and are retained in the
ER. The misfolded protein is not degraded, and accumulates in the ER (The
endoplasmic reticulum serves many general functions, including the folding of
protein molecules and the transport of synthesized proteins to the Golgi apparatus) of
hepatocytes resulting in liver damage.
Myloid accumulation
Amyloid refers to the abnormal fibrous, extracellular, protein deposits found in
organs and tissues. Amyloid is insoluble and is structurall domina ed b -sheet
structure. Amyloid fibrils are formed by normally soluble proteins, which assemble
to form insoluble fibers that are resistant to degradation. One of the hallmarks of
Alzheimer's disease is the accumulation of amyloid plaques between nerve cells
(neurons) in the brain.
Huntington's disease
Huntingtons disease is a neurodegenerative disorder that is caused by an unstable
expansion of a CAG repeats within the coding region of the HTT gene. The HTT
gene, which encodes huntingtin, a protein of unknown function, is located on the
human chromosome 4. One region of the HTT gene contains a particular DNA
segment known as a CAG trinucleotide repeat. This segment is made up of a series of
three DNA building blocks (cytosine, adenine, and guanine) that appear multiple
times in a row. The disease-causing mutation is a CAG repeat expansion located
within exon of the HD gene. The CAG repeat is translated into a polyQ stretch. As a
result, the translated protein huntingtin contains disease-causing expansions of
glutamines (polyQ) that make it prone to misfold and aggregate.

Terminology:
Nucleus - membrane closed organelle found in eukaryotic cells. Cell nuclei contain most
of the cell's genetic material. The nucleus also contains the nucleolus. It's main functions
are many, it is called the cells ''control center''
Nucleolus - the largest structure in the nucleus of eukaryotic cells. The nucleolus is made
of proteins and RNA, it's main function is to synthesize ribosome.
Nucleoid - The nucleoid (meaning nucleus-like) is an irregularly shaped region within
the cell of a prokaryote that contains all or most of the genetic material, called genophore.
In contrast to the nucleus of a eukaryotic cell, it is not surrounded by a nuclear
membrane.
Organelle - Organelles have a wide range of responsibilities that include everything from
producing hormones and enzymes to providing energy.
Centrosome - An organelle that serves as the main microtubule organizing center of the
animal cell. Centrosomes are composed of two centrioles. The centrioles organize the
microtubules assembly during cell division.
Chromatin - Chromatin consists of DNA complexed with histone proteins inside the
nucleus.
Genome - Complete set of DNA, including all of its genes.
DNA methylation - DNA methylation is a process by which methyl groups are added to
the DNA molecule. Methylation can change the activity of a DNA segment without
changing the sequence. When located in a gene promoter, DNA methylation typically
acts to repress gene transcription.

Colloquium 2
Lecture 5 - Epigenetics
Epigenetics is the study of heritable phenotype changes that do not involve alterations in
the DNA sequence. Epigenetics are about how the DNA is read, and how it is expressed.
Epigenetic traits is the regulating system for the body to decide which genes that are
expressed or repressed.
Epigenetic regulation in eukaryotes
Epigenetic events in the eukaryotic organism provides:
Precise and stable control of gene expression
Genomic stability (Genomic instability is a high frequency of mutations within
the genome)
Epigenetic traits have a crucial role in genomic stability - silencing of centromeres,
telomeres and transposable elements ensure:
The correct attachment of microtubules to centromeres
Reduce excessive recombination between repetitive elements
Prevents transposition of transposable elements
There are three categories of signals that are involved in the establishment of a stably
heritable epigenetic state.
1) The Epigenator
Changes in the environment of the cell trigger epigenetic changes of the cell. The
environment signal is considered as the Epigenator. Examples of Epigenator signals are:
temperature variations, metabolites, differentiation signals. Once an Epigenator signal is
received, it leads to activation of the initiator.
2) The Epigenetic Initiator
The initiator translates the Epigenator signal. The initiator then identifies the location on
a chromosome where epigenetic marks will be established. Epigenetic Initiator is;
DNA binding proteins
The DNA-binding proteins are DNA sequence specific, and they bind through non-
covalent interactions between an a-helix in the DNA binding protein domain and
atoms on the edges of the bases within a DNA major groove; DNA sugar-phosphate
backbone atoms. Atoms in the DNA minor groove also contribute to binding.
Non-coding RNA (ncRNA)
Epigenetic related ncRNAS are the short ncRNA and long ncRNA. They're function
is to regulate gene expression at the transcriptional and post-transcriptional level.
1. Short ncRNAs (<30 nts)
- microRNA (miRNA) - bind to a specific target mRNA with a
complementary sequence to induce; cleavage, degradation, block translation.
- Short interfering RNA (siRNA) - Mediate post-transcriptional gene silencing
which results in mRNA degradation through inducing heterochromatin
formation by binding RISC, which promotes histone methylation.

- piwi-interacting RNA (piRNA) - chromatin regulation and suppression of
transposon activity in germline and somatic cells.
2. Long ncRNA (>200 nt)
The long ncRNAs form complex with chromatin-modifying proteins and
recruit their catalytic activity to specific sites in the genome, the result is
modification of chromatin state and influenced gene expression.
3) The Epigenetic Maintaining
The Maintainer sustains the epigenetic trait but is not sufficient to initiate it. This signal
involves many different pathways, including:
Histone modifying enzymes
Histone modification is a covalent post-translational modification (PTM) to histone
proteines. PTM work together to regulate the chromatin structure, which affects
biological processes including gene expression, DNA repair and chromosome
condensation.
Histones consist of a globular histone core and has two terminal tails (N-terminal, C-
terminal tail), the majority of histone PTMs occurs on the N-terminal tail. Due to their
chemical properties, these epigenetic modifications alter the condensation of the
chromatin and there the accessibility of the DNA to the transcriptional machinery
(The more condense chromatin structure -> less accessible, The less condense
chromatin structure -> more accessible)
- Methylation, the addition of a methyl group to a molecule. Histone methylation can
either increase or decrease transcription of genes. Enzymes regulating histone
methylation (add - uncoiling - more accessible): HMT. Enzymes reulating histone
demethylation (remove - condense - less accessible): HDM.
- Phosphorylation, the addition of a phosphate group to a molecule, most commonly
associated with transcriptional activation, as the negative charge of phosphate group
creates repulsive force between the histone and negatively charged DNA, it still is
reversible though. Proteins regulating the histone phosphorylation is protein kinases
(PK) (add - uncoiling - more accessible). Proteins regulating the histone
dephosphorylation is protein phosphatases (PP).
- Acetylation, the addition of a acetyl group to a molecule, the acetyl group
neutralizes the charge leading to decreased affinity between histone tail and DNA.
Enzymes regulating histone acetylation (add - uncoiling - more accessible): HAT.
Enzymes regulating histone deacetylases (remove - condense - less accessible):
HDAC.
- Ubiquitylation, the addition of ubiquitin protein to the histone core proteins H2A
and H2B. Ubiquitination on H2A is considered repressive, H2B ubiquitination has
been associated with both active and repressed genes.
- Sumoylation, the addition of Small-Ubiquitin-like-Modifier protein to a substrate
protein, added to their targets by specific ligases. Histone sumoylation is a mark of
transcriptional repression.
DNA modifying enzymes/DNA-methylation
DNA-methylation is a process by which methyl groups are added to the DNA
molecule. Methylation can change the activity of a DNA segment without changing

the sequence. When located on the CpG-island (gene promoter), DNA methylation
acts to repress gene expression. CpG sites are methylated by one of three enzymes
called DNA methyltransferases (DNMTs).
DNA methylation is also found at sites other than CpG sequences, the process is
referred to as non-CpG cytosine methylation. This have been identified at a high level
in stem cells indicating that loss of this form of methylation may be critical in the
differentiation.
Histone variants
We have five major histone families; H1, H2A, H2B, H3, H4. By which the first is
linker histone, and the four later are core histones (histone octamer - nucleosome).
Proteins that substitute the core histones are called histone variants, these are coded
by different genes. Histone variants have specific expression, localization and
distribution pattern. Functionally they affect chromatin remodeling and post-
transcriptional modifications.
Nucleosome remodeling
Nucleosome remodeling refers to the change in the structure of chromatin, a process
which requires ATP energy input. Nucleosome remodeling is carried out by enzymes
ATPase. This enzyme action may lead to:
- Complete or partial disassembly of nucleosomes
- The exchange of core histones for variants
- The assembly of nucleosomes
Epigenomics
The epigenomics are changes in the whole genome, this can lead to the genes reprogram
and is expressed differently than what the DNA is signaling. The epigenome signals can
dominate what has been inherited by the parents. The epigenome is influenced by the
environment (diets, toxins, hormones, environmental factors and more).
Imbalance of gene expression -
An allel is a variant form of a given gene. New alleles are created by mutations. The
diploid set of human chromosomes carry alleles on given places (loci). Because we have
chromosome couples, we have place for 2 alleles of which one is paternal and other
maternal. Usually these two copies are expressed at the same level. When the gene
expression levels varies (the ratio is not 1 to 1) from each allele it is referred to as allelic
imbalance.
Monoallelic expression - only one of the two copes of a gene is active, while the other is
silent. It has four types:
Somatic rearrangement
Somatic rearrangement is changes in the DNA organization that produces only one
functional gene copy, this choice of gene copy is random. The mechanism that causes
the monoallelic expression is - cutting and pasting of DNA sequences to rearrange
genes in somatic cells to generate enormous antibody diversity.
Random allelic silencing or activation

Random allelic silencing or activation is expression from only one gene copy at
chromosomal localization (locus), due to different epigenetic changes. The process
has been classically studied in gene families in the nervous and immune systems.
Genomic imprinting
As we are given two copies of genes, some genes can only be expressed based on if
it's either paternal or maternal. Genomic imprinting is when one copy of a gene is
silenced due to its parental origin. The actual process of imprinting is done during the
gametogenesis.
X chromosome inactivation
Females carry two copies of the gene rich X-chromosome, this can potentially result
in a toxic double dose of X-linked genes - to correct this gene dosage imbalance the
mechanisms vary with species. Mammalian females have the X-chromosome
inactivation mechanism. X chromosome inactivation is the epigenetic silencing of X
chromosome linked genes on one female chromosome, the x chromosome that is
silenced is of random choice.
RNA plays an important role in X inactivation, especially XIST. Xist is induced by
doubled escape gene product, and following; silencing of genes on the inactive X
chromosome take place. The X chromosome is inactivated by methylation of
cytosine, H4 histone hypo-acetylation and other histone modification.
In XY males, the lower dose of the escape gene product is insufficient to induce Xist
and to silence X-linked genes.
Epigenetic/Epigenomics in human health and disease
Dutch Hunger Winter
Prenatal conditions can influence peoples health across their lifetime. During the dutch
hunger winter famine children born to mothers starving for the last few months of
pregnancy were smaller in size, and stayed small all their lives. The children born to
mothers that starved for the first months were abnormal sized, and had higher obesity
rates than normal. These abnormal sized children were found to have less methylation
(gene silencing) of the insulin growth factor which codes for growth, this increased the
gene expression for this factor and possibly lead to an abnormal size of the children and
later on even obesity.
Cancer epigenomics
Classically in cancer tissues DNA methylation (gene silencing) is reduced globally.
Hypomethylating oncogene promoters results in reduced defence against repetitive
sequences leading to genome instability and chromosome structural changes.
Lecture 6 - Human genome variation
BASE TERMINOLOGY
Allele - The different forms (alternative forms) of a gene or a DNA sequence. These
alleles are placed on a certain chromosomal location (locus) of a gene. You have one
maternal gene, one paternal gene - these can be of different(hetero)/same(homo) allele.
Homozygote - An individual in whom the two alleles at a locus are the same.
Heterozygote - An individual who has two different alleles at a locus.

Hemizygote - An individual who only one gene (unpaired genes, for example: X-linked
genes in males)
There are different types of alleles; wild-type or variant.
Wild-type allele
The wild-type allele is a single prevailing (rådande, kraftigare), it is the most common
allele
Variant allele
An allele that differs from the wild-type allele is called a variant. They differ due to
permanent changes in the nucleotide sequence or arrangement in DNA sequence.
The nature of genetic variation
Genetic variation is a term used to describe the variation in the DNA sequence in each of
our genomes. Genetic variation is what makes us all unique, whether in terms of hair
colour, skin colour or even the shape of our faces.
Mutation
All genetic variation originates from the process known as mutation, which lead to
alteration in human genome. Mutations can either be due to a spontaneous process or
induced.
Spontaneous process of mutation
The most common source of spontaneous mutations is due to errors in DNA
replication. Some examples are: Replication slippage, errors in DNA reparation,
errors in recombination, errors in cell division.
Induced process of mutation
The induced process of mutation is due to environmental factors. These can be:
- Physical - ionized radiation, UV - This can result in deletion when the modified
strand is copied. Deletion is a mutation where a part of the chromosome or sequence
is lost during DNA replication.
- Chemical - oxidative stress, aromatic amines, deaaminating agents etc. Deamination
is the removal of an aminogroup from a molecule, this can result in point mutations
(single nucleotide change) when the template strand is copied.
- Biological - transposons, viruses.
Alteration in human genome can be observed in different level
Chromosomal aberration
Chromosomal aberrations is any change in the number (numerical aberrations) and
structure of chromosomes (structural aberrations). There are two maintypes of
chromosomal aberration:
1) Numerical aberrations -
Aneuploidy -
Loss or gain of one chromosome, for example a human cell having 45 or 47
chromosomes instead of the usual 46.

Aneuploidy originates during cell division when the chromosomes do no separate
properly between the two cells (missegregation)
Euploidy -
Euploidy is a condition when a cell or an organism has one or more than one
complete set of chromosomes. For example if a human would contain 69
chromosomes (3n) then it would be considered euploid.
Numerical aberrations result in failure in meiotic (formation of gametes) or mitotic
(after conception) cell division.
2) Structural aberrations -
Structural aberrations occur due to loss of genetic material, or a rearrangement in the
location of the genetic material. Structural rearrangements are defined as balanced if
the complete chromosomal set is still present though rearranged, and defined as
unbalanced if there is additional or missing information. Types of structural
aberrations:
- Deletion (Unbalanced)
Chromosome break and subsequent loss of genetic material.
Terminal deletion: A single break leading to a loss that includes the chromosomes tip
is called terminal deletion.
Interstitial deletion: An interstitial deletion results when two breaks occur and the
material between the break is lost
A deletion sometimes occur at both tips of a chromosome this can form a circular
chromosome as both ends fuse and form a ring chromosome.
- Duplication (Unbalanced)
A portion of the chromosome is duplicated, this can arise from unequal cross over.
Duplication results in extra genetic material.
- Inversion (Balanced)
A portion of the chromosome has broken off, turned upside down and reattached,
therefore the genetic material is inverted. If the inversion includes the centromere, it
is called pericentric inversion. Inversions that do not include the centromere are
called paracentric inversions.
- Translocation (Balanced)
Translocation is a interchange of genetic material between non-homologous
chromosomes. There are two main types of translocations but they both result in
derivative chromosomes:
a) Reciprocal transition - Segments from two non-homologous chromosomes are
exchanged.
b) Robertsonian transition - An entire chromosome has attached to another at the
centromere.
DNA sequence alterations
DNA alterations occur in the DNA base sequence. DNA alterations commonly refer
to a single gene alteration or alteration of a non-coding sequence. There are two
maintypes of DNA sequence alterations:

1) Base substitution
Base substitution is the process of which one base is substituted for another. The
effect of this can vary:
a) Silent mutation - The change in the nucleotide base has no outward effect, it only
alters the genetic code by synonymous replacement of one amino acid.
b) Missense - Missense refers to a base substitution which changes the amino acid
coded for by the affected codon. It alters the genetic code by a NON-synonymous
replacement of one amino acid.
c) Non-sense - This refers to a base substitution in which the changed nucleotide
transforms the codon into a stop codon (UAA, UAG, UGA) in the mRNA.
2) Deletion/Insertion
When a nucleotide is wrongly inserted/deleted from a codon, the effect can be drastic
in form of a frameshift mutation. A nucleotide that is wrongly inserted/delted can
affect every codon in a genetic sequence by throwing the entire three by three codon
out of order. Examples of deletion/insertion:
- Insertion of mobile elements - Insertion of ALU and LINE repeats can cause
frameshift mutations.
- Dinamic mutation - Amplification of a simple nucleotide repeat
Alterations of DNA sequences can result in allelic variants. The consequences allelic
variants in a functional level are varying:
Gain of function - Allelic variant lead to completely new protein product (with
new function) or overexpression of product or inappropriate expression (wrong
time, wrong place etc.)
Loss of function - loss of gene product activity.
Dominant negative - The abnormal protein product interferes with the normal
protein product and inhibits its functioning.
Interpretation of DNA sequences alteration:
Pathogenic - Causing/capable of causing disease
Like pathogenic - Alterations with strong evidence in favor of disease
Benign - Alterations with strong evidence against pathogenesis
Likely benign - Alterations that are likely to be benign
Uncertain significance - Alterations with limiting or/and conflicting evidence
regarding pathogenesis
Alteration of DNA sequence VS. Chromosomal aberrations
Changes
Chromosomal aberration is any change in
the number and structure of chromosomes
DNA alterations occur in the DNA base
sequence
Scale

Chromosomal aberration can include many
gene alterations
DNA alterations commonly refer to a single
gene alteration or alteration of non-coding
sequence
Damage
Damages due to chromosomal aberration are
large scale compared to DNA alteration
Nucleotide damage is small in scale
compared to chromosomal aberration
Lecture 7 - Cell division
BACKGROUND STRUCTURE
The cytoskeleton is a network of filaments and tubules that extend throughout a cell,
through the cytoplasm. The cytoskeleton ensure ability of a eukaryotic cell to:
Resist deformation - maintain cell's shape
Transport intracellular cargo (e.g. vesicles)
Change shape during movement (e.g. cell division, organelle migration)
Assists in the transportation of communication signals between cells
The cytoskeleton consists of:
1) Microtubules -
The largest type of filament, composed of the protein tubulin. Tubulin is composed of a-
tubulin and B-tubulin assembles into protofilaments. A single microtubule contains 10-15
protofilaments that wind together and form a hollow cylinder. The structure of the
microtubules allows them to grow (polymerization) or shrink (depolymerization) in size.
There are three types of microtubules with vital function during cell division:
a) Astral microtubule - Ensures correct positioning and orientation of mitotic spindle.
b) Kinetochore microtubule - Attaches to kinetochore of chromatids
c) Interpolar microtubule - Extend from spindle pole across the equator
2) Microfilament -
The smallest type of filament, composed of the protein actin.
3) Intermediate filament -

The intermediate filaments are composed of different protein subunits. There are various
intermediate filaments depending on which cell they are present in, e.g. neurofilaments in
neurons.
Centrosome -
In cells the minus end of microtubules are anchored structures called microtubule
organizing centers (MTOC). The primary MTOC in a cell = centrosome. Consists of two
centrioles (constructed of microtubules), duplicated during S-phase.
Motorprotein -
Motorproteins are a class of molecular motors that can move along the cytoplasm of the
cell. They use the energy derived from ATP hydrolysis to generate movement and force.
There are three motor proteins involved in cell division:
a) Kinesin -
Moves along microtubules to pull organelles toward the cell membrane.
b) Dynein -
Pulls cellular component inward, towards the nucleus.
c) Myosin -
Interact with actin to perform muscle contractions, involved in cytokinesis, endocytosis,
and exocytosis.
Mitosis: Cell divison
Mitosis is the somatic cell division that gives rise to two genetically identical daughter
cells. Mitosis is refers to the actual nuclear division (karyokinesis), and is followed by
cytokinesis (division of the cell cytoplasm) which is the second part of the M-phase.
Regulation of mitosis -
The key cell cycle regulation proteins are cyclin-dependent kinases (CDK). CDK
ensures accurate cell cycle progression.
Kinase - adds phosphate group to molecule.
Polo-like-kinases (Plks) and Aurora kinases, are major regulators of centromsome
function, spindle assembly, chromosome segregation and cytokinesis.
Mitosis consists of five phases:
1. Prophase
The chromosome condense into compact structures
Condensins attach to chromosomes that coil the chromosomes into highly compact
forms - > H1 histone phosphorylation and attachment of condensins are mediated by
Cdk1, H3 histone phosphorylation is mediated by Aurora B kinase.
The highly compact structures are held together to form sister chromatids, the
sister chromatids are held together by rings formed by cohesin.
The nuclear envelope breaks down to form a number of small vesicles. The
nucleolus disintegrates. Transcription and synthesis stops.
Formation of the mitotic spindle begins (Plks and Aurora kinases)
The centrosomes gradually move to take up positions at the poles of the cell (Plks
and Aurora kinases)

2. Prometaphase
The chromosomes are completely attached to the mitotic spindle. Spindle fibres
are binded to kinetochore.
The chromosomes, led by their centromeres, start migrate to the equatorial plane
of the cell. This region is known as the metaphase plate.
3. Metaphase
The chromosomes line up along the metaphase plate
The spindle fibres attach to the centromeres of the sisterchromatids. These fibres
act to separate the sister chromatids. This is mediated by the APC/C complex
(anaphase promoting complex) as it helps cohesin degrade, also cyclin A and B
are involved.
(The mitotic spindle checkpoint can be activated in case of mistake in mitotic spindle
assembly)
4. Anaphase
Each chromosomes sister chromatids separate and move to opposite poles of the
cell. The separated sister chromatids are now referred to as daughter
chromosomes.
Astral microtubules -> pulls poles further apart, Kinetochore microtubules ->
shorten and draw chromosomes toward the spindle poles, Interpolar microtubules
-> Slide past eachother, exerting additional pull on chromosomes
5. Telophase
The chromosomes arrive at the cell poles
The mitotic spindle disassembles
The vesicles assemble around the two sets of chromosomes
Phosphatases dephosphorylate the lamins at each end of the cell. This
dephosphorylation results in the formation of a new nuclear membrane around
each group of chromosomes.
Cytokinesis -
Cytokinesis is the physical process that finally splits the parent cells into two identical
daughter cells. The signal for the start of cytokinesis is dephosphorylation of proteins,
which are targets for Cdks.
The cell membrane pinches in at the cell equator, forming a cleft called the cleavage
furrow. The action of a contractile ring of overlapping actin and myosin filaments forms
cleavage furrow.
Biological role of mitosis -
Growth of the organism -
Mitosis help in increasing the number of cells in a living organism thereby playing a
significant role in the growth of a living organism
Repair/Replacement -

Mitosis helps in the production of identical copies of cells and thus helps in repairing
the damaged tissue or replacing the worn-out cells.
Asexual reproduction
Genetic stability -
Mitosis helps in preserving and maintaining the genetic stability of a particular
population.
Regulation of mitosis -
The key cell cycle regulation proteins are cyclin-dependent kinases (CDK). CDK
ensures accurate cell cycle progression.
Kinase - adds phosphate group to molecule.
Polo-like-kinases (Plks) and Aurora kinases, are major regulators of centromsome
function, spindle assembly, chromosome segregation and cytokinesis.
Meiosis
Meiosis is the germ cell division that gives rise to gametes (sperm and egg). The process
by which diploid cells give rise to haploid cells. It involves two rounds of cell division,
with only one round of DNA replication (Meiosis 1)
Meiosis 1 -
Consists of four stages:
1. Prophase 1
The prophase of meiosis 1 is a complicated process with several defined stages:
a) Leptotene (Greek for thin threads) - Chromosomes begin to condense.
b) Zygotene (Greek for paired threads) - Chromosomes become closely paired.
Homologous chromosomes begin to along their entire length, forming synapsis (pairing
of homologous chromosomes). Chromosomes are held together by a synaptonemal
complex.
c) Pachytene (Greek for thick threads) - synapsis is completed and there is a genetic
exchange of genetic material between homologous pairs, this exchange of genetic
material is called crossing over. The pairs appear as bivalent.
PICTURE
d) Diplotene (Greek for two threads) - after recombination, the synaptonemal complex
begins to break down. Homologous chromosomes begin to separate but remain attached
by the chiasmata.
e) Diakinesis (Greek for moving through) - chromosomes condense and separate until
terminal chiasmata only connect the two chromosomes.
Homologous recombination:
Homologous recombination - process in which DNA molecules are broken and the
fragments are rejoined in new combinations. Genetic variability is produced by genetic
recombination through the process of crossing over. Crossing over is ensured by
homologous recombination. Involves double stranded breaks (DSB) followed by
homologous reparation mediated by recombination complex. The formation of DSBs is
catalysed by highly conserved proteins with topoisomerase activity. In the absence of
recombination, chromosomes often fail to align properly for the first meiotic division –

as the result there is high incidence of chromosomal loss, called nondisjunction. A
failure in homologous recombination is often reflected in poor fertility.
2. Metaphase 1
Nuclear membrane disappears
A spindle forms
The paired chromosomes align themselves on the equatorial plane with their
centromeres oriented toward different poles. The orientation is random.
3. Anaphase 1
The homologous are pulled apart and move to opposite ends of the cell. Their
respective centromeres with the attached sister chromatids are drawn to opposite
poles of the cell. This process is termed disjunction. Each maternal and paternal
chromosome in a homologous pair segregates randomly into a daughter cell in
meiosis 1.
4. Telophase 1
The two haploid sets of chromosomes have grouped at opposite sites of the cell.
Cytokinesis -
The cell divides into two haploid daughter cells and enters meiotic interphase.
Meiosis 2
The second meiotic division is similar to an ordinary mitosis except that the chromosome
number of the cell entering meiosis II is haploid. There is no DNA replication before the
next division
Lecture 8 - The cell cycle
The cell cycle describes the life cycle of a cell, the periods between sucessive division of
a cell. There are differences between cell cycle in prokaryotes and eukaryotes.
Cell cycle in prokaryotes -
The cell division process of prokaryotes, called binary fission, is a less complicated and
much quicker process than cell division in eukaryotes. Because of the speed of bacterial
cell division, populations of bacteria can grow very rapidly. The normal life cycle of a
bacterial cell involves:
Replication phase -
The replication of the bacterial genome occurs, results in formation of new
chromosome
Division phase -
The segregation of daughter chromosome and other cellular components into
daughter cells. This process is initiated by FtsZ proteins which assemble in a ring and
lead to formation of septum.
Interval phase -
The period between division and the initiation of chromosome replication.

Cell cycle in eukaryotes -
1. Interphase -
G1 phase
The gap phase after cell division. First phase of interphase. Cell grows physically
larger, protein synthesis occurs and organelles are copied. The cells conducts series of
checks before entering the S phase.
S phase
The DNA synthesis phase. Starts with replication of DNA and finishes when the
amount of DNA in the cell is doubled.
G2 phase
The gap phase after S-phase and before Mitosis. The cells grows more. The cell
conducts series of checks before entering the M-phase.
2. M-phase (mitosis)
The cell divides it's copied DNA and cytoplasm to make two new cells.
3. G0 - the resting phase
The two daughter cells produced can either undergo immediate round of cell division or
the cell can slowly re-enter the cell cycle or not at all. The cells that slowly re-enter/not at
all, enter a state called G0 phase, the resting phase. Cells can be either be quiescent (re-
enter the cell cycle) or senescent (do not re-enter the cell cycle).
Regulation of cell cycle in eukaryotes -
CDKs
The key cell cycle regulation proteins are cyclin-dependent kinases (CDKs) to ensure
accurate cell cycle progression. Each CDK associate with different cyclin (to activate
kinases activity) and the associated cyclin determines which proteins are
phosphorylated by a particular-cyclin-CDK complex.
There are three main cyclin-CDK complexes:
- G1 cyclin-CDK
Reacts to exogenous signals. Regulates phosphorylation in a way that allows the
expression of genes required for DNA synthesis. Important for transition from G1 to
S-phase.
- S-phase cyclin-CDK
Promotes DNA synthesis, targets are helicase and polymerases.
- Mitotic cyclin-CDK
Important for transition from G2 to M-phase, activates APC ligase. Regulates G2/M
checkpoint.
Regulation of cell cycle by check points -
Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity and
fidelity (nogrannhet) of the major events of the cell cycle. Functions:
Growth to appropriate cell size
The replication
Integrity of the chromosomes

Accurate segregation of mitosis
- G1/restriction checkpoint
Functions: Checks cell size, nutrients, growth factors and DNA damage by environmental
factors.
If damage is found; G1-arrest or/and G0 phase
- S phase checkpoint
Function: Responds to DNA damage by spontaneous mutations. Checks the stabilization
of DNA replication fork and responds to DNA damage.
If damage is found; The replication is put on hold until problem is resolved.
- G2 phase checkpoint
Function: DNA damage checkpoint that serves to prevent the cell from entering M-phase
with genomic DNA damage.
If damage is found: The proteins that sense DNA damage signal the cell-cycle machinery,
the response is p53 that activates gene expression for specific proteins to induce
apoptosis.
- M-phase checkpoint
Function: Check for the mitotic spindle assembly - prevents separation of duplicated
chromosomes until each chromosome is properly attached to the spindle apparatus.
If damage is found: Arrest anaphase in case of mistake.
Regulation of DNA replication by DNA repair mechanisms -
DNA repair processes exist in both prokaryotic and eukaryotic organisms. Control of
DNA repair is closely tied to regulation of the cell cycle - during the cell cycle,
checkpoint mechanisms ensure that a cell's DNA is intact both before and after DNA
replication. There are various DNA repair mechanisms:
DNA polymerase and its ''proofreading'' mechanism. The mispaired bases are
replaced by proofreading mechanism, extra nucleotides are inserted. After the
detection of a misplaced base, a catalytic site of the DNA polymerase known
as the exonuclease digests the mispaired nucleotides from the growing chain.
Mismatch repair (MMR) -
Repairs DNA replication errors. Removes base mismatches and small
insertion/deletion loops. The MMR repair mechanism is strand-specific as it
distinguishes the newly synthesized strand and the template strand.
Human pathology and MMR: Lynch syndrome - mutations cause impair MMA
function, this condition gives increased risk for cancers.
Nucleotide excision repair (NER) -

Repairs DNA damages inducted by environmental factors - radiation e.g. excision
of UV light inducted DNA damage. Two repair sub-pathways exist: TC-NER
(transcriptional active DNA) and GG-NER (global genomic)
Human pathology and NER: Xeroderma pigmentosum - mutations cause impaired
NER mechanism, NER is not able to effectively repair DNA damages inducted by
radiation.
Base excision repair (BER)
Removes damaged bases in DNA sequence. Responsible for removing small, non-
helix-distorting errors. BER can repair: oxidized bases, alkylated bases,
deaminated bases, inappropriately incorporated uracil and single strand DNA
breaks.
BER is initiated by DNA glycosylase that recognizes and removes the damaged
base. Two pathways exist: Short (removes on base lesions) and long (removes 2-
10 base lesions)
Recombination repair
Environmental factors can cause double-stranded breaks in DNA. There are two
types of recombination repair, the choice of mechanism depends on cell cycle
stage:
1) non-homologous end join - ( more active G1)
The two broken ends are put back together without DNA template, this typically
includes the loss or some addition of a few nucleotides at the break site.
2) homologous end join - (more active S and G2-phase)
Information from a homologous chromosome is used as a template to replace the
damaged region of the broken chromosome.
Cell aging and apoptosis -
Cells go through a natural life cycle which includes growth, maturity, and death. This
natural life cycle is regulated by a number of factors, and the disruption of the cycle is
involved in many disease states. For example, cancer cells do not die the way normal
cells do at the end of their life cycle.
Cell aging -
Cellular senescence is the irreversible arrest of cell proliferation. The two main pathways
involved are - p53/p21 that lead to growth arrest and p16/pRB that lead to
heterochromatin.
Apoptosis -
Form of cell death, also known as programmed cell death. Apoptosis leads to:
fragmentation of the DNA, shrinkage of the cytoplasm, membrane changes and cell death

without damage to neighboring cells. It is initiated by protease caspases, once it has been
initiated reversible.
Lecture 9 - Gametogenesis
Gametogenesis is the development of haploid sex cells. It matures the oocyte (egg cell)
and sperm cell in diploid organisms by meiosis.
Origin of gametes -
a) The formation of gametes or sex cells start at 4 weeks of pregnancy
Sex cell precursors - PGC (primordial germ cell) arise in the yolk sac which is an
extra embryonic structure.
b) After 4-6 weeks of pregnancy PGC start to migrate from yolk sac to genital
ridge and develop into bisexual gonads.
c) Depending on if a embryo is genetically male (46, XY) or female (46 XX)
there will be different processes for sex differentiation:
Male - XY - medula will develop into embryonic testis - and the PGC cells will
develop into spermatogonia in embryonic testis. The surrounding cells will
develop into supporting cells, called sertoli cells in testis.
The same activation mechanisms are used for both sexes. If SRY-gene (located on
Y chromosome and autosomes) is expressed it will lead to the activation of
SOX9-gene which promotes differentiation of testis and at the same time
suppresses genes and proteins that would be important for ovary differentiation.
Female - XX - cortical part will develop into the embryonic ovaries - and the PGC
cells will develop oogonia in embryonic ovaries. The surrounding cells will
develop into supporting cells, called follicula cells in ovaries.
The same activation mechanisms are used for both sexes. If SRY-gene (located on
Y chromosome and autosomes) does not expressed it will not activate SOX9-
gene, which activates the genes involved in determining the ovary differentiation.
Epigenetic regulation of gametogenesis -
Because germ cell genes, but not somatic cell genes, are passed on to the next generation
there is a difference in the managing of imprinted marks on these genes. At first the
imprinted marks of the zygot must be all erased. As the somatic cells in the embryo are
not passed on, the imprinted marks must be put back as received from parents. The
imprinted marks present on gametes of the developing embryo however need to be reset
according to the sex of the developing embryo.
a) PCG undergoes genome-wide-demethylation (the epigenetic marks are erased)
b) During migration to the bisexual gonad, some of the marks are put back
c) When PCG has migrated to the bisexual gonad, there is a second wave of DNA
demethylation which erases methylation marks of imprinted genes in the PCG genome

d) Later during fetal life there is a remethylation of germ cell genome depending on the
sex of the embryo. Paternal imprint -> before meiosis, and at time of birth. Maternal
imprint -> Occurs during ovulation
Oogenesis
The process of which the female gametes (ägg cell) are created
Periods of oogenesis:
Proliferation - (prenatal period = before birth) (mitosis occurs)
3rd month of embryonic development
From PCG -> oogonia
Primary oogonia -> Secondary oogonia
Growth -
4-6 months of embryonic development
Secondary oogonia -> primary oocytes
By the 5th month degeneration begins
Maturation - (Meiosis 1, Meiosis 2)
7-8th month of embryonic development (start of meiosis 1, mitosis stops)
Meiosis 1 is initiated but then stops at diplotene, there is a meiosis arrest at birth.
Puberty - > (arrest still at meiosis 1)
The rest of the primary oocytes created are reduced further. The follicles develop to
primary follicles. At this point (puberty), the meiosis 1 is resumed. As the primary
follicles have started developing into secondary follicles, every 5-15 months the
primary oocyte matures. And only one primary oocyte actually matures until the end,
finishing the meiosis 1 -> which finishes with the cytokinesis which results in cell
division -> The cytokinesis is unequal due to one cell receiving all the cytoplasm (the
secondary oocyte) with half the chromosome number and one (polar body) with less
cytoplasm but half the chromosome number (polar body degenerates). The secondary
oocyte finishes meiosis 1, and then enters the fallopian tube where it continues the
meiosis 2, where it matures and a second polar body is created. The second arrest
happens in meiosis 2, metaphase 2, and can only be completed if fertilized. Scenarios:
1) Absence of fertilization - Secondary oocyte stays in meiosis 2, metaphase 2. The
mature follicle shrinks, corpus luteus formes (promotes development of next oocyte)
2) Fertilization occurs - The sperm cell binds to egg cell in the fallopian tube, triggers
Secondary oocyte continues and finishes meiosis 2. And the zygot is created.
Spermatogenesis -
Spermatogenesis starts at puberty and continues til death, the process occurs in the testis.
The spermatogenesis can be divided into two prenatal and postnatal periods.
1) Prenatal -
PGC becomes spermatogonia in embryonic testies
2) Postnatal -

At the time of birth sex cells are located in sex cords that include immature sperm cells
and supporting cells. The sex cords will then develop into testies, and the immiture germ
cells will develop into sperm cells meanwhile the supporting cells develop into sertoli
cells.
Sertoli cells -
Sertoli cells have similar function as the follicule cells in oogenesis, they mainly guide
the sex cells.
Functions:
Surround developing sperm cells – tight junctions; hemato-testicular barrier
Secrete proteins and fluids, which means they have trophic function: feed sex
cells
Secrete growth and other factors, needed for development of the sex cells
Synchronize the events of spermatogenesis
Spermatogenesis can be divided into 4 periods.
Proliferation -
Begins in embryonic life and continues throughout life
Mitosis occurs 9-10 times -> produces millions of cells
From PGC -> Primary (A) spermatogonia (2n)
Primary (A) spermatogonia -> Secondary (B) spermatogonia (2n)
Growth -
Begins in puberty
Secondary (B) spermatogonia grows -> Primary spermatocytes (2n) (mitosis)
Maturation:
Primary spermatocytes (2n) goes through meiosis 1, and the cell division creates
two secondary spermatocytes (n) each having half of the chromosomes of the
primary spermatocytes.
The secondary spermatocytes (n) then go through the meiosis 2 and differentiate
into two spermatids (n) per secondary spermatocyte -> 4 spermatids, each having
a haploid chromosome set.
Spermiogenesis -
The spermatids then differentiate into spermatozoa through a process called
spermiogenesis. Steps:
Formation of an acrosome
The acrosome is an organelle that develops over the anterior half of the head in the
spermatozoa. The acrosome contains enzymes that allow sperm to penetrate oocyte.
Nuclear morphogenesis
Changes the shape of the head from round to oval, and makes it more dense through
chromatin condensation by the help of sperm specific histones.
Formation of tail structures

The tail structure is an elongation of microtubules from the distal centriole pressing
out of the cytomembrane.
Rearrangement of organelles
Most of the organelles are lost, but mitochondria rearranges and to midpiece part. The
mitochondria is the source of energy for the spermatozoa to move.
Shedding most of the cytoplasm
Abnormal sperm -
Abnormal sperm can affect the functions of the sperm, following are examples of
abnormal sperm
Change in shape and size of head
Multiple head
Double body
Abnormal midpiece
Sperm production syndromes -
Azoosperma - no sperm in semen (infertile)
Oligozoosperma - low sperm count (<20 million per ml)
Sertioli cell only syndrome - No sex cells, just sertoli cells
Kartegener syndrome - Heredity condition -> affects mobility of sperm flagella
Spermagenesis VS. Oogenesis
Spermagenesis Oogenesis
Begins at puberty, ends at death Begins before birth, ends at menopause
Encompass 4 periods: proliferation, growth,
maturation, spermiogenesis
Encompasses 3 periods: proliferation,
growth, maturation

Only primary spermatocyte forms 4 sperm
cells
One primary oocyte forms one egg and
polar bodies
Billions of sperm cells are produced at a
time
One oocyte matures montly
Cytokinesis is not completed until the end
of spermatogenesis
Nuclear division takes place at very end of
oogenesis
Molecular reactions: when egg is reached
a) Acrosome undergoes exocytosis - sperm releases digestive enzymes into zona
pellucida
b) The sperm migrates through the coat of the follicle cells (corona radiata) and binds to a
receptor mole cule in zona pellucida of the egg
c) ZP3 mediates sperm-specific egg binding
d) With the help of acrosomal reaction the sperm reaches the egg, and a membrane
protein (ADAM) of the sperm binds to a receptor (ZP2) on the egg membrane
e) The plasma membrane fuse making it possible for sperm cell to enter the egg
Terms:
Phenotype - An organisms physical appearance or a physical trait, that varies between
individuals. For example: size or eye color.
Epigenetic trait - a stably heritable phenotype resulting from changes in a chromosome
without alterations in the DNA sequence.
Epigenomics - Epigenetic changes in the whole genome.
Genome - The entire genetic material of an organism.
Stem cell - The primal (ursprunglig) cell that other specialized cells can derive from, they
can differentiate into other types of cells. This is through turning on/off certain genes in
the stem cell that code for certain functions.
Gonad - Ovaries or testicles
Zygot - The cell built when two gametes (egg and sperm) are fused, the zygot is diploid
Somatic cell - the body's cells except gametes/sex cells

Molecular Biology, Script - 3rd
Colloquium
Molecular transport
The endomembrane system
The endomembrane system is a group of membranes and organelles within eukaryotic cells that work
together to modify, package and transport lipids and proteins.
The endomembrane system includes:
Nuclear envelope
The nuclear envelope is made of two lipid bilayer membranes which surround the nucleus which
encases the genetic material of the cell.
The endoplasmic reticulum (ER)
The endoplasmic reticulum is a type of organelle found in eukaryotic cells that forms an
interconnected network of structures known as cisternae. It is continuous with the nuclear
envelope. The ER plays a key role in the modification of proteins and synthesis of lipids.
There are two types of ER: rough ER (outer face studded with ribosomes) and smooth ER.
Rough ER Smooth ER
Site for protein synthesis Site for lipid, glycogen and
steroid synthesis
Protein translocation,
folding and transport
Metabolism of
carbohydrates
Glycosylation (addition of
sugar groups)
Detoxification function
Disulfide bond formation,
to stabilize tertiary and
quaternary structures of
proteins
Major storage and release
site of calcium ions
The Golgi apparatus
The Golgi apparatus is an organelle with various functions:
Protein sorting and export through secretory pathways
Protein glycosylation
Lipid and polysaccharide metabolism and transport
Formation of lysosomes
Lysosomes
The lysosome is an organelle that contains digestive enzymes and acts as the organelle-recycling
facility of an animal cell. It breaks down old unnecessary structures, so their molecules can be
reused.

Plasma membrane
The cell membrane/plasma membrane defines the borders of the cell and allows interaction of the cell
with its environment in a controlled way. To perform these roles the plasma membrane needs:
Lipids
The plasma membrane needs lipids, which make a semi-permeable barrier
between the cell and its environment. All the lipid molecules in cell
membranes are amphipathic, meaning they have a hydrophilic group and
a hydrophobic tail. This amphipathic nature causes the tail groups to self-
associate into a bilayer with the polar head groups oriented toward water. There are different types
of lipids:
Phosphoglycerides The head group is phosphate and an alcohol (hydrophilic), the tail group is a
long fatty acid tail (hydrophobic).
Sphingolipids The head group is an amino alcohol (hydrophilic), the tail group is a fatty acid tail
(hydrophobic)
Glycosphingolipids Belong to group of sphingolipids. Same structure with attached
carbohydrate.
Steroids - Class of lipids, which have structures totally different from the other classes of lipids. The
basic structure is a four-ring hydrocarbon.
Cholesterol – Belong to group of steroids.
Proteins
The plasma membrane needs proteins, which are involved in cross-membrane transport and cell
communication. There are three main categories: Integral membrane proteins, Lipid-anchored
membrane proteins and Peripheral membrane proteins.
Semi permeable barrier =
Permeable only to certain
small molecules

Integral membrane proteins – Integral membrane proteins are integrated into the membrane: The
portions of an integral membrane protein found inside the membrane are hydrophobic, while those
that are exposed to the cytosolic and exoplasmic domains have hydrophilic exterior surfaces.
Lipid-anchored membrane proteins – Lipid-anchored membrane proteins are located on the surface
of the cell membrane, they are bound covalently to one or more lipid molecules within the cell
membrane.
Peripheral membrane proteins – Peripheral membrane proteins are either localized on the
exoplasmic- or cytosolic face, they do not interact with hydrophobic core of the phospholipid
bilayer.
Carbohydrates
Carbohydrates are the third major component of plasma membranes. They are found on the outside
(exoplasmic face) surface of cells and are bound either to proteins or lipids. They are able to interact
with components of the extracellular matrix, growth factors and antibodies.
Transport of small molecules
Small molecules such as ions, water and small organic molecules are imported to the cell. Cells also
make and alter many small molecules by series of different chemical reactions. Small molecules have
various of functions in the cells:
Precursors for synthesis of macromolecules
Store and distribute energy for cellular processes

Signaling function
The internal composition of the cell is maintained because the plasma membrane is selectively
permeable to small molecules. The selection of molecules is based on: size, charge and solubility of the
molecule.
Impermeable Permeable Slightly permeable
Large uncharged polar
molecules; Glucose,
fructose
Gases; CO2, N2, O2
Ions; K+
, Ca2+
Small uncharged polar
molecules; Ethanol
Small uncharged polar
molecules; Water
Charged polar
molecules; Amino acids,
ATP, proteins, nucleic
acids
There are different types of membrane transport:
Passive (simple) diffusion
Passive transport is the movement of molecules across a cell membrane, without the need of energy
input. The molecule dissolves in the phospholipid bilayer, diffuses
across it, and then dissolves in the aqueous solution at the other side
of the membrane. The direction of transport is determined by the
relative concentrations of the molecule inside and outside of the cell,
and the transport reactions are spontaneous.
Facilitated diffusion
Facilitated diffusion is the passage of molecules across a cell
membrane through specific transport proteins and requires no energy
input. Facilitated diffusion allow polar and charged molecules (such as
carbohydrates, amino acids, nucleosides, and ions) to cross the
plasma membrane.
Diffusion is the movement of
particles down their gradient. A
gradient is any imbalance in
concentration and moving down a
gradient just means that the particle
is trying to be evenly distributed
everywhere.

a) Carrier proteins
Membrane carrier protein that binds to a solute and transports it across hydrophobic regions of
the cell membrane, by undergoing conformational changes (carrier proteins can change their
shape to move a target molecule from one side of the membrane to the other). All carrier
proteins are transmembrane proteins, as they extend all the way across the membrane.
Carrier proteins are uniporters as they transport a single-type of molecule down its
concentration gradient.
b) Channel proteins
Channel proteins form a hydrophilic passageway through opening pores in the membrane,
allowing small molecules of the appropriate size and charge to move through the lipid bilayer.
They transport water or specific types of ions and hydrophilic small molecules down their
concentration gradients.
Channel proteins can either be non-gated (channels are opened most of time, or gated (open
only in response to specific chemical or electrical signals).
Aquaporins –
Form of channel proteins, that form pores in the membrane mainly facilitating water
transport between cells.
Ion channels –
Form of channel proteins, that form pores that allow ions to pass through the channel
pore. Ion channels are highly selective, because of the narrow pores in the channel
restrict size and charge.
Gated ion channels are not permanently open, this function is based on type of gating:
Ligand-gated channels – Open in response to the binding of signaling molecules.
Voltage-gated channels – Changes in the electric potential across the plasma
membrane.
Active transport
Active transport is the movement of molecules across a membrane from a region of their lower
concentration to a region of their higher concentration against the concentration gradient. Active
transport requires cellular energy to achieve this movement. There are different types of active
transport:
Ion pumps Transmembrane protein that moves ions across a plasma membrane against their
concentration gradient through active transport, these transporters convert energy from various
sources.
ATP-powered pumps ATPases that use the energy of hydrolysis to move ions or small
molecules.
Cotransport/secondary active transport
Cotransporters are membrane transport proteins that use
the energy stored in an electrochemical gradient to
transport two substances across a membrane.
There are two types of co-transport proteins; symport
(both substances are transported in the same direction)
and antiport (the substances are transported in opposite
direction).
Electrochemical gradients = As atoms
and molecules can carry electrical
charges, there may be a difference in
charge (electrical gradient) across a
plasma membrane.

Ion channel defect and human pathology
Disease, symptoms: Cystic fibrosis, a genetic disease characterized by the buildup of thick mucus that
can damage man of he bod s organs. This abnormal mucus can clog airways leading to severe
problems with breathing and bacterial infections in the lungs.
Causes: Mutations in the CFTR gene cause cystic fibrosis. The CFTR gene provides instruction for making
cyclic AMP regulated chloride ion channel that transports chloride ions into and out of cells, chloride has
an important function in cells such as controlling the movement of water in tissues which is necessary
to produce thin, freely flowing mucus. Mutations in the CFTR gene disrupt the function of the chloride,
as a results mucus that is unusually thick is produced.
Transport of macromolecules
A macromolecule is a large complex molecule, such as nucleic acids, proteins, carbohydrates and lipids.
Eukaryotic cells can take up macromolecules and particles from the surrounding by a distinct process
named endocytosis.
BASIC PROCESS: First, the plasma membrane of the cell invaginates (folds inward), forming a pocket
around the target particle or particles. The pocket then pinches off with the help of specialized proteins,
leaving the particle trapped in in a newly created vesicle or vacuole inside the cell.
There are various endocytosis pathways that can be subdivided in the following categories:
Phagocytosis
Phagocytosis (literally, cell eating) is a form of endocytosis in which large particles such as
microorganisms and dead cells are transported into the cell via large endocytic vesicles called
phagosomes. Professional phagocytes include: macrophages, neutrophils and dendritic cells.
a) Binding of particle to receptors on the surface of cell triggers the extension of pseudopodia
(the cell surface is extended)
b) The fusion of pseudopodia forms phagosome. A phagosome is a vesicle formed around a
particle engulfed by a phagocyte via phagocytosis.
c) The phagosome then fuses with lysosomes, producing phagolysosomes.
Pinocytosis
Type of endocytosis in which soluble materials are taken up from the environment and
incorporated into vesicles for digestion.
Receptor-mediated endocytosis
A form of selective uptake of macromolecules in which receptor proteins on the cell surface are
used to capture a specific target molecule. The receptors cluster in regions of the plasma
membrane called coated pins (clathrin-coated pins). When the receptors bind to their specific
target molecule, endocytosis is triggered, and the receptors and their attached molecules are
taken into the cell in a vesicle.
An example of receptor-mediated endocytosis is the uptake of cholesterol. Cholesterol is
transported through the bloodstream in form of lipoprotein particles, the most common of
which is called LDL.

LDL receptor and human pathology
Disease, symptoms: Familial Hypercholestrolemia, condition characterized by very high levels of
cholesterol in the blood.
Causes: Mutations in the LDLR genes cause hypercholesterolemia. The LDLR gene provides instructions
for making a protein called LDL receptor. This type of receptor binds to LDLs, which are the primary
carriers of cholesterol in the blood. These receptors play a critical role in regulating cholesterol levels, by
removing LDLs from the bloodstream.
Exocytosis
Exocytosis is the process by which molecules are release outside of the cell in membrane-bound vesicles
that fuse with the plasma membrane. Exocytosis can be divided into two subcategories:
Constitutive exocytosis
Constitutive exocytosis is carried out by all cells and serves to transfer molecules from the Golgi
network to the outer surface of the cell. Most proteins are transported directly to the cell
surface by the nonselective constitutive secretory pathway (does not require particular signal).
Regulated secretion
Regulated secretion occurs in response to specific conditions, signals or biochemical triggers,
and is the process underlying the release of cytokines, hormones, neurotransmitters and other
small signaling molecules.
a) Secretory proteins are packed into secretory vesicles
b) Secretory vesicle fuses with the plasma membrane, contents are discharged from the cell by
exocytosis, and its membrane becomes part of the plasma membrane
c) Membrane components from secretory vesicles are removed from the surface by
endocytosis.
The balance between the forward and retrograde flows of membrane needs to be precisely
regulated.
Diversity of vesicles
Clathrin coated vesicles mediate transport from the plasma membrane and the Golgi network
COPI- and COPII-coated vesicles mediate transport between the ER and the Golgi apparatus and
between Golgi cisternae

Before the vesicle fuses with a target membrane, the coat is discarded, to allow the two cytosolic
membrane surfaces to interact directly and fuse.
Role of plasma membrane in molecular transport
Separates the contents of the cell from its outside environment and it regulates what enters and
exits the cell.
The cell membrane is primarily composed of proteins and lipids. While lipids help to give
membranes their flexibility and proteins monitor and maintain the cell's chemical climate and
assist in the transfer of molecules across the membrane
The lipid bilayer is semi-permeable, which allows only selected molecules to diffuse across the
membrane.
Signalling
Cell adhesion and the extracellular matrix
Cell communication, between cells and the extra cellular matrix, is critical to the development and
function of multicellular organisms. Cells communicate through chemical and mechanical signals.
Cell adhesion is the process by which cells can interact and attach to neighboring cells through
molecules on the cell surface. There are various ways of cell adhesion:
1. Direct contact
Direct contact is when cells are bound directly to one another by cell-cell junctions. There are
various forms of cell-cell junctions:
Tight junctions are a form of cell-cell junction that seal adjacent epithelial cells together,
this functions as a barrier preventing the passage of most dissolved molecules from one
side of the epithelial sheet to the other.
Gap junctions are a form of cell-cell junction that allow ions and small molecules to pass
from the cytoplasm of one cell to the cytoplasm of the next
Adherens junctions (anchoring junction) are a form of cell junction in which the
cytoplasmic face of the plasma membrane is attached to actin filaments
Desmosomes (anchoring junction) are a form of cell junction in which dense plaques of
protein are linked with intermediate filaments
2. Indirect contact
Hemidesmosome (anchoring junction) is a specialized cell junction between an epithelial
cell and the underlying extracellular matrix
Extracellular matrix (ECM)
The extracellular matrix is a scaffold (stöd/byggställning) of proteins and
carbohydrates located around cells. The complex network is composed
of polysaccharides and proteins secreted by cells, these structures are connected through protein-
protein interactions and protein-carbohydrate adhesions. The extracellular matrix serves as a structural
element in tissues, ensures mechanical properties, cell communication, cell migration and cell
differentiation.
Important structures of the ECM
Extracellular means located outside of
the cell

The most abundant proteins of the ECM are Collagens and Elastin. In the ECM some collagen proteins
are attached to glycosaminoglycans (GAGs) forming proteoglycan. The ECM is connected to
surrounding cells, fibronectin (glycoprotein) is a key connector in this interaction, this interaction helps
ECM regulate cell adhesion, migration and differentiation. Another glycoprotein helping the interaction
between cells is the laminins, mediating interactions between cells via cell surface receptors. The
glycocalyx is a layer/covering of glycoproteins and sugars that surrounds the extracellular face (outside)
of the plasma membrane, the composition of the glycocalyx influences properties of the cell such as:
adhesion, cell-cell recognition and signaling.
Signal transduction
Extracellular signaling molecules function within an organism to control:
Metabolic processes within cells
The growth and differentiation of tissues
The synthesis and secretion of proteins
The composition of intracellular and extracellular fluids
The communication by extracellular signals involves six steps:
Synthesis of the signaling molecule
Release of the signaling molecule
Transport to the target cell
Detection of the signal by a specific receptor protein
Cellular response to the signal
Removal of the signal
Extracellular signaling molecules are synthesized and released by signaling cells. Signaling molecules
operate over various distances:
Paracrine Signals from one cell can act on nearby cells, e.g. growth factors
Endocrine Signals from one cell can act on distant cells, e.g. sending signal throughout the
whole body by secreting hormones into the bloodstream
Autocrine – Signals act on the signaling cell itself, e.g. certain types of T lymphocytes respond to
antigenic stimulation by synthesizing a growth factor that drives their own proliferation, and
thereby amplifying the immune response.

Signaling across gap junctions direct diffusion of ions and small molecules between adjacent
cells, e.g. epithelial tissue.
Neuronal the signaling cells (neurons) release the chemical ligands (neurotransmitters), a long
distance from the cell body. Physical extension of the cell itself (axon) spans the long distance to
a specific target so the ligand is released in a space very close to the target cell (synapse), e.g.
neurons and muscle cells.
Contact-dependent signaling, Juxtacrine – Juxtacrine is a type of cell signaling that requires
close contact. In this type of signaling, a cell places a specific ligand on the surface of its
membrane, and subsequently another cell binds it with an appropriate cell surface receptor.
Receptors
Signaling molecules produce a specific response only in target cells with receptors that are specific to
the signaling molecules. The receptors can be activated by:
Binding of secreted or membrane-bound molecules
Changes in the concentration of metabolite
Physical interaction
The signaling molecule act as a ligand (primary messenger), which binds to a structurally
complementary site on the extracellular or membrane-spanning domains of the receptor. Receptors
and ligands come in closely matched pairs, with a receptor recognizing just one (or a few) specific
ligands, and a ligand binding to just one (or a few) target receptors. Binding of a ligand to a receptor
changes its shape or activity, allowing it to transmit a signal or directly produce a change inside of the
cell. The receptors can be divided into two categories: Intracellular receptors and cell membrane
receptors.
1. Intracellular receptors –
Receptor proteins found on the inside of the cell. The ligands of the intracellular receptors are
hydrophobic messengers, since they must be able to cross the plasma membrane in order to
reach their receptors. The intracellular receptors regulate expression of specific genes.

2. Cell-membrane receptors
Cell-membrane receptors bind to ligands on the outside surface of the cell. In this type of
signaling, the ligand does not need to cross the plasma membrane and therefore it the ligands
are hydrophilic messengers.
A ligand binds its receptor through several specific weak non-covalent bonds by fitting into a specific
binding site.
Affinity is the measure of the strength of attraction between a receptor and its ligand. High receptor
affinity low concentrations of a ligand will result in binding of most of the corresponding receptors.
Low receptor affinity high concentration of the ligand is required for most receptors to be occupied.
Prolonged exposure to a ligand may lead to desensitization of the cell, this can also occur by removal of
ligand (receptor mediated endocytosis).
Binding of a ligand to its receptor (ligand-receptor interaction) causes a conformational change
(anpassning) in the receptor that induces specific cellular responses. Second messengers then relay the
signal from one location to another (e.g. from plasma membrane to nucleus). There are three major
classes of second messengers:
Hydrophilic – cAMP, cGMP, IP3, Ca+2
(calcium)
Hydrophobic – DAG, PIP3
Gaseous – NO, CO

G protein-coupled receptors (GPCRs)
G protein-coupled receptors are receptors that are coupled with G proteins (epinephrine, glucagon,
serotonin, vasopressin, ACTH, adenosine). G proteins are specialized proteins that can bind GTP and
GDP. G proteins that are associated with GPCRs are trimeric, meaning they have three subunits: 1) G
alpha 2) Beta 3) Gamma. Activated G proteins bind to enzymes or other proteins (effectors) and alter
the target proteins activity. Inactivated G proteins are bound to GDP.
GPCRs signaling pathway
1) Ligand binds receptor (GPCR)
2) This binding triggers our GCPR to undergo a conformational (anpassning) change
3) Due to this conformational change, the G alpha subunit exchanges GDP for GTP
4) This exchange causes our G alpha subunit to dissociate from the other subunits. The G proteins
subunits can now regulate (activate or inhibit) target proteins. These target proteins can be
enzymes that produce second messengers or ion channels that let ions be second messengers.
5) Once this G protein subunit activates a target protein, this target protein can relay
(vidarebefodra) a signal via a second messenger
6) GTP is hydrolyzed to GDP, once this once happens the G subunits become inactive
GPCRs and human pathology
Disease, symptoms: Retinitis pigmentosa, a group of related eye disorders that cause progressive
(gradvis) vision loss. These disorders affect the retina (light-sensitive tissue at the back of the eye),
vision loss occurs as the light-sensing cells of the retina gradually deteriorate.
Causes: The genes associated with retinitis pigmentosa play roles in the structure and function of light
receptor cells in the retina. The retina contains two types of photoreceptors: rods and cons. Mutations
in any of the genes responsible for the disease lead to gradual loss of rods and cones in the retina.

Receptors with intrinsic or associated enzymatic activity
Enzyme linked receptors receive signals from the environment and instruct the cell to do certain things,
in addition to this ability they also function as enzymes. The enzyme linked receptors act as enzymes
once the extracellular binding site has bound a ligand, which induces the intracellular site to function as
an enzyme.
The most common enzyme linked receptors are receptor tyrosine kinases (RTKs), they regulate cell
growth (bind to EGF epidermal growth factor), differentiation and survival.
1) Ligand (e.g. insulin, EGF, FGF, neurotrophies) binds, this causes neighboring RTKs to associate
forming a cross-linked dimer
2) This cross-linking activates the tyrosine (located on intracellular site), which causes
phosphorylation (process: cross-phosphorylation)
3) Once cross-phosphorylated the intracellular site serve as docking platforms for different cellular
proteins involved in signal transduction
4) As different proteins are attached, this allows activation of multiple different intracellular
signaling pathways. (1) Ras MAP kinase pathway; (2) IP3/DAG pathway; (3) PIP3 kinase
pathway.
RTKs and human pathology
Disease, symptoms: Achondroplasia is a form of short-limbed dwarfism.
Causes: Mutations in the FGFR3 gene cause achondroplasia. The FGFR3 gene provides instructions for
making a protein that is involved in the development and maintenance of bone and brain tissue. In
achondroplasia the cartilage (flexible tissue) is converted into bone. «Gain-of-function» causes further
inhibition of chondrocyte growth, by constitutive activation of the receptor which causing premature
conversion of the growth plate into bone, leading to skeletal defects
Ion channel receptors
Ligand-gated-ion channels are ion channels that open or close in response to the binding of a ligand (e.g.
neurotransmitters, cGMP, physical factors, IP3). Receptor structure is four or five subunits with a
homologous segment in each subunit lining the ion channel.
1) Once a specific ligand binds to the receptor, it can control the opening or closing of the ion
channel by altering the entire protein conformation
2) Once the channel opens it will let ions (e.g. calcium, sodium, potassium, chlorine) move through
the channel
3) This movement will cause a change in the electrical properties of a cell -> the extracellular
ligand signaling is converted to intracellular electrical signal (response)
4) Elevation of cytosolic calcium ions
Ligand-gated ion channels and human pathology
Disease, symptoms: Congenita myasthenic syndrome is a group of conditions characterized by muscle
weakness.
Causes: Mutations in CHRNA1 gene is common cause, the CHRNA1 gene codes for the acetylcholine
receptor (AChR). Specially from abnormally brief opening and activity of the channel.

Intracellular receptors
Intracellular receptors are receptors located inside the cell rather than on its cell membrane. for steroid
hormones, thyroid hormones, retinoids and vitamin D. The receptor contains three domains: 1)
Transcription-activating domain 2) DNA-binding domain 3) Ligand-binding domain. The receptor-ligand
complex binds in the promoter region of the target genes: in case of hormones it is called a hormone
response element (HRE)
Intracellular receptors and human pathology
Disease, symptoms: Androgen insensitivity syndrome, disorder that affect people that are genetically
male, as their bodies are unable to respond to certain male sex hormones (androgens) they may have
mostly female external sex characteristics or signs of both.
Cause: Mutations in the AR gene cause androgen insensitivity syndrome. AR is a ligand-dependent
nuclear receptor that functions as a DNA-binding transcription factor, leading to the regulation of
biological processes such as development, cellular proliferation, differentiation and apoptosis. The AR
gene provides instructions for making a protein called an androgen receptor. Androgen receptors allow
cells to respond to androgens, which are hormones such as testosterone, that direct male sexual
development.
Molecular biology in medicine
Molecular medicine promotes the understand of normal body functioning and disease pathogenesis at
the cellular and molecular level. It allows researchers and physicians to apply this knowledge to disease
diagnosis, treatment, prognosis and prevention.
In vitro methods in diagnostics –
In vitro are studies performed with microorganisms, cell or biological molecules outside of their normal
biological context (Opposite: in vivo -> conducted inside animals/whole plats). Studies of in vitro are
conducted using components of an organism that have been isolated from their usual biological
surrounding.
Omics is a technique used in molecular medicine that falls under the
category of in vitro methods. Omics is a field of study in biology ending
in -omics:
Genomics Study of the genome of organisms
PCR – Polymerase chain reaction
PCR is considered the basic method of molecular biology used to amplify (make more copies
of) DNA regions of known sequences. This method is applied in genetic research, medicine-
diagnostics, microbiology & virology, mycology (muscles) & parasitology, forensic science
(juridisk) and environmental science. Workflow in PCR:
(1) Blood sample
(2) Extraction of DNA
(3) PCR
(4) Gel electrophoresis
Gel electrophoresis –
Genome is an organisms complete set
of DNA

Gel electrophoresis is a method used for separation and analysis of macromolecules (DNA,
RNA and proteins) based on their size and charge. Electrophoresis involves running a
current through a gel containing the molecules of interest. Based on their size and charge,
the molecules will travel through the gel in different directions or at different speeds,
allowing them to be separated from one another. There are different types of gel used for
this method, most common: Agarose- and Polyacrylamide gel.
1. Agarose gel electrophoresis –
Agarose is a complex sugar derived from seaweed. It is made of chains of
interlinked sugars. The agarose is polymerized by cooling. Agarose runs
horizontally. Resolution (separation) 50 – 30 000 bp of DNA (different
concentration of gel). Agarose gel have lower resolving power of DNA but
have greater range of separation.
2. Polyacrylamide gel electrophoresis (PAG) –
Polyacrylamide gel is made of a chemical crosslink of acrylamide and bis-
acrylamide. PAG runs vertically. PAG is usually used for proteins and have
high resolution (separation) for shorter DNA fragments of 5 -500bp length.
Gel electrophoresis is often used in combination with PCR to visualize the results of PCR.
Diagnostics of Cystic fibrosis (CF) using PCR and electrophoresis
Disease, symptoms: Cystic fibrosis (CF), is caused by allelic variants in CFTR gene, the most
common allelic variant: p. Phe508del or p. Gly551Asp
Cause: The p. Phe508del allelic variant is caused by a 3 nt deletion (in frame) in the CFTR
gene which leads to missing Phe in position 508. This mutation leads to defective channel
processing and gating problems. The p. Gly551Asp allelic variant is caused by single
nucleotide exchange.
A laboratory technique that can be used to detect allelic variants (such as CF) is the
Restriction fragment length polymorphism (RFLP) method. Restriction enzymes are DNA-
cutting enzymes, a restriction enzyme recognizes and cuts DNA only at a particular sequence
of nucleotides. Workflow in RFLP:
(1) Blood sample
(2) Isolation of DNA
(3) PCR
(4) Incubation with restriction enzyme
(5) Gel electrophoresis
(6) RFLP analysis
Diagnostics of LCHADD using RFLP and electrophoresis –
Disease, symptoms: LCHADD is a condition that prevents the body from converting certain
fats to energy.

Cause: It is caused by allelic variants in HADHA gene. The most common allelic variant is
1528G>C which results in Glu substitution with Gln in 510 position.
Sanger (direct) sequencing -
The sanger sequencing is a method used to detect sequences of DNA fragments that are up
to 800-900 bp long. Workflow in Sanger sequencing:
(1)
(2) Blood sample
(3) Isolation of DNA
(4) PCR
(5) Sequencing reaction
(6) Capillary electrophoresis
(7) Data analysis
Next generation sequencing (NGS) –
Next generation sequencing is a DNA sequencing technology, of which an entire human
genome can be sequenced within a single day. Through targeted sequencing it allows the
identification of disease-causing allelic variants for diagnosis of pathological conditions.
Pros Cons
Takes less time and money in
comparison to Sanger sequencing
Is still considered expensive in
many labs
Provides a deeper coverage of
genomic regions, and higher-
throughput than Sanger
sequencing
Inaccurate sequencing of repeated
nucleotide regions can lead to
sequence errors this affects the
quality of data for diagnostics
Data analysis can be time-
consuming and require special
knowledge
Transcriptomics
T an c ip omic a e echni e ed o d an o gani m an c ip ome he m of all i
RNA transcripts)
Reverse transcription for PCR (RT-PCR) –
A variant of PCR technique used in molecular biology used to detect mRNA and
quantitation.
Quantitative PCR (qPCR) –
Quantitative PCR combines PCR amplification and detection into a single step. Fluorescent
dyes are used to label PCR products during thermal cycling. Instruments measure the
accumulation of fluorescent signal during the exponential phase of the reaction in real-time.

Workflow in transcriptomics:
Metabolomics and proteomics
Metabolomics is the study of chemical processes including metabolites. Workflow in
metabolomics:
Proteomics is the large-scale study of proteins. Workflow in proteomics:

Precision (personalized) medicine
Precision medicine refers to the tailoring of medical treatment to the individual characteristics of each
patient. Examples of precision medicine are the following:
Personalized treatment of CF –
When treating CF, doctors can tailor therapies to patients based their genetic makeup. Depending on
the individual patient symptoms they are offered treatment. Patients with CF can be offered:
Patient 1) p. Phe508del is an allelic variant that can cause CF. The allelic variant is caused due to a
deletion that produces a CFTR protein with a folding defect, this affects the chloride transport.
Treatment: Restoring chloride transport to p.Phe508del CFTR require correction (Lumacaftor) of cellular
misprocessing to increase the amount of functional mutated CFTR.
Patient 2) G551D is an allelic variant that can cause CF. The allelic variant that causes a channel opening
defect. Treatment: Potentiation (Ivacaftor) help to further increase channel opening in of most class III
(gating) allelic variants.
Biotechnology
Biotechnology can be described as the use of living organisms or biological processes for the purpose of
developing useful agricultural, industrial or medical products.
Examples of biopharmaceuticals produced by biotechnology processes are:
Antibiotics
Blood clotting factors
Hormones
Cytokines
Growth factors
Enzymes
Vaccines
Current biotechnological processes essentially involve five different groups of organisms:
Bacteria
Fungi
Plants
Insects
Mammalians
An example of a biotechnological method is Recombinant DNA technology, which is the joining
together of DNA molecules from two different species that are inserted into a host organism to
produce new genetic combinations that are of value to science, medicine, agriculture, and industry.
The first licensed drug produced using recombinant DNA technology is human insulin. Recombinant
human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae.

Mol bio script 1 (2) (4).pdf

More Related Content

Similar to Mol bio script 1 (2) (4).pdf (20)

More from Vamshi962726 (20)

Recently uploaded (20)

Mol bio script 1 (2) (4).pdf