CURSO 0

MASTER EN GESTIN Y DESARROLLO DE

TECNOLOGAS BIOMDICAS

Schedule
LUNES

19:00

MIERCOLES

JUEVES

Curso 0.
Curso 0.
Curso 0.
Economia y
Biologa
Economa y
Gestion
Molecular
Gestin
Aula: 7.2.J04 Aula: 2.3.B03 Aula: 2.3.A04

15:30

17:15

MARTES

VIERNES
Curso 0.
Economa y
Gestin
Aula: 7.2.J04

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Curso 0.
Curso 0.
Curso 0.
Biologa
Economia y
Biologa
Economa y Curso 0. Biologia
Molecular
Gestin
Molecular
Gestin
Celular MOOC
Aula: 7.2.J04 Aula: 7.2.J04 Aula: 2.3.B03 Aula: 2.3.A04

Curso 0.
Biologa
Celular MOOC

Curso 0.
Biologa
Celular
MOOC

Curso 0.
Economa y
Gestin
Aula: 2.3.A04

CURSO MOOC: INTRODUCCIN A LA MEDICINA REGENERATIVA Y LA INGENIERA TISULAR

Registrarse en: https://spoc.uc3m.es/

Goal
To learn/review the most relevant celular and molecular biology concepts in
order to be able to fully understand the course lessons.

Bibliography

Essential Cell Biology. Bruce Alberts et al. Garland Science. At the library.

Biomedical Engineering. W. M. Saltzman. Cambridge Univ. Press. At the library

ACCESS TO MOOC

CURSO 0
LECTURE 1
INTRODUCTION TO MOLECULAR BIOLOGY
NUCLEIC ACIDS STRUCTURE AND PACKAGING

Our life is maintained by molecular

network systems

Molecular network
system in a cell
(From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)

Molecular Biology
The study of the structure, function and
composition of the biologically most relevant
molecules (HGP)

Molecule: Defined and ordered

aggregation of atoms, the smallest part in
which you can divide a substance that
conserves all its properties.
The atomic composition is practically the
same in all living organisms: H 63%, O 24%,
C 10%, N 1.4%, P 0.2%, S<0.1% + traces of
Mg, Ca, Zn, K, Mn

Figure 2-26 Essential Cell Biology ( Garland Science 2010)

BIOPOLYMERS

All living cells are made up of four classes of biological molecules: carbohydrates,
lipids, proteins and nucleic acids
Within cells, small molecules are joined together to form larger molecules (polymers)

Figure 2-15 Essential Cell Biology ( Garland Science 2010)

Table 2-1 Essential Cell Biology ( Garland Science 2010)

Biological macromolecules are large polymers. The primary

structure (linear) is maintained by covalent bonds. Higher
order structures (2-D, 3-D) require flexibility and are
provided by weaker bonds.

Hydrophobic and hydrophilic forces

Hydrophobic interactions describe the
relations between water
and hydrophobes (low water-soluble
molecules). Hydrophobes are
nonpolar molecules and usually have
a long chain of carbons that do not
interact with water molecules.

Hydrophilic interactions are

interactions between water and other
molecules such that the other
molecules are attracted to water

Eukaryotic Cell

Eukaryotic cells are found in

animals, plants, fungi and protists
cell;
Cell with a true nucleus, where
the genetic material is surrounded
by a membrane;
Eukaryotic genome is more
complex than that of prokaryotes
and distributed among multiple
chromosomes;
Eukaryotic DNA is linear;
Eukaryotic DNA is complexed
with proteins called histones;
Numerous membrane-bound
organelles;
Complex internal structure;
Cell division by mitosis.

Prokaryotic Cell

Unicellular organisms, found

in all environments. These
include bacteria and archaea;
Without a nucleus; no nuclear
membrane (genetic material
dispersed throughout
cytoplasm;
No membrane-bound
organelles;
Cell contains only one circular
DNA molecule contained in the
cytoplasm;
DNA is naked (no histone);
Simple internal structure; and
Cell division by simple binary
fission.

DNA structure
Primary structure
Secondary structure
Tertiary structure
a polymer of deoxyribo nucleotides
found in the nucleus of euakaryotic cells, mitochondria and chloroplasts
carries the genetic information

Components of a nucleotide
Sugar

Components of a nucleotide
Base

Pyrimidines

Purines

Structure of a nucleotide
Phospate

Base

Phosphate
Sugar
X=H: DNA
X=OH: RNA

Nucleoside
Nucleotide

The primary structure of DNA is the sequence

5
5 end

Phosphodiester
linkage

DNA: The transforming principle

1869: Miescher discovers nuclein in the pus of chirurgical gauzes
1928: Griffith: Spanish influenza, studies in pneumoccocus gave first evidence
1943: Avery McCarthy and McLeod finished Griffiths experiments

DNA: The transforming principle

1952: Hershey-Chase experiments, bacterial viruses carry the progeny information in DNA.
Previously in 1940, Chagraff had discovered the rules of Chagraff : %[A]=%[T] and %[G]=%[C]
in all living organisms

Nature 171: 737-738 April 1953

Watson JD and Crick
FC (1953) Molecular
Structure of Nucleic
Acids: A Structure
for Deoxyribose
Nucleic Acid.
1962 Nobel Prize
awarded to three
men Watson, Crick
and Wilkins

A mention to Rosalind Franklin

The secondary structure of

DNA
Two anti-parallel polynucleotide
chains wound around the same axis.
Sugar-phosphate chains wrap
around the periphery.
Bases (A, T, C and G) occupy the
core, forming complementary A T
and G C Watson-Crick base pairs.
Polarity matters!

Two hydrogen bonds between A:T pairs

Three hydrogen bonds between C: G paired

Normally hydrated DNA: B-form DNA

Helical sense: right handed

Base pairs: almost

perpendicular to the helix
axis; 3.4 apart
One turn of the helix: 36
; ~10.4 base pairs

Minor groove: 12 across

Major groove: 22 across

The DNA double helix is held together mainly by hydrogen bonding and
base stacking

Base Stacking
The bases in DNA are planar and have a

tendency to "stack". Pi stacking (also called

stacking) refers to attractive, noncovalent
interactions between aromatic rings, since they
contain pi bonds.

Major stacking forces:

hydrophobic interaction

van der Waals forces.

information stored using a code of four symbols.

A book written with an alphabet of four letters

Figure 5-8 Essential Cell Biology ( Garland Science 2010)

Structural forms of DNA

Tertiary structure: DNA Packaging

The Room Problem:
How to pack 2m in a few m

DNA: 2m
Pack. Factor:
10^5-10^6

Figure 5-19 Essential Cell Biology ( Garland Science 2010)

Cell Cycle

Figure 5-25 Essential Cell Biology ( Garland Science 2010)

Histones: positively
charged basic proteins
Nucleosome: 147 bp

Interphase

Metaphase

Structure of nucleosome core

Beads nucleosomes (8 histones)

Chromatin: DNA+proteins (50% each)

Figure 5-21 Essential Cell Biology ( Garland Science 2010)

Compaction of DNA in a eukaryotic chromosome

Supercoil = coil over coil

Metaphase chromosomes

Species/ Number of Chromosomes

Species

Number of chromosomes

Human

46

Mouse

40

Rat

42

Fruit flies

Bacteria

Ploidy is the number of sets of chromosomes in a cell.

Diploid (2n): has two copies of the genetic information
Haploid (n): a cell that has half the usual number of chromosomes or just one copy
of the genetic information

LOS CROMOSOMAS
El nmero de cromosomas de cada especie es fijo
En la especie humana hay 23 parejas de cromosomas. 22
parejas son AUTOSOMAS y la pareja 23 son los CROMOSOMAS
SEXUALES.
Un cromosoma de cada pareja proviene de cada uno de los
progenitores (CROMOSOMAS HOMLOGOS)

Locus: place where a gene maps

Pattern of
bands/interbands

telomer
centromer

telomer
Figure 5-11 Essential Cell Biology ( Garland Science 2010)

The Gene

The gene is a segment within a very long strand of DNA.

Genes are the basic units of hereditary.
Genes located on chromosome on its place or locus.
Allele: a variant of the DNA sequence at a given locus.
Each allele inherited from a different parent.

EXPRESION DE LOS ALELOS

Como existen dos cromosomas homlogos, se
combinan dos alelos para cada carcter.
Estos alelos pueden ser iguales o diferentes
Si son iguales, los individuos son
HOMOZIGOTOS para el carcter

Si son diferentes, son HETEROZIGOTOS

para el carcter

RELACIONES ENTRE LOS ALELOS

ALELOS DOMINANTES Y RECESIVOS
Cuando dos alelos van juntos en cromosomas
homlogos, siempre se expresar uno de ellos que ser
el ALELO DOMINANTE. El otro que no se manifiesta es
el ALELO RECESIVO.
Al alelo Dominante se le asigna la letra
mayscula del GEN.
A
Al alelo Recesivo se le asigna la letra
minscula del gen.
a

RELACIN ENTRE LOS ALELOS

HERENCIA INTERMEDIA
Cuando los alelos que van juntos en
cromosomas homlogos dominan por igual. De
modo que el individuo manifiesta una mezcla de
los dos alelos

RELACIN ENTRE LOS ALELOS

HERENCIA CODOMINANTE
Cuando los alelos que van juntos en
cromosomas homlogos dominan por igual. De
modo que el individuo manifiesta los dos alelos
a la vez, pero sin mezclarse.

LOS GRUPOS SANGUINEOS

GENOTIPO Y FENOTIPO
GENOTIPO.- Es el conjunto de alelos que tiene un
individuo para los diferentes caracteres.
FENOTIPO.- La manifestacin del genotipo. Es decir
el carcter que se manifiesta.

Genotipo: A a (heterocigotico)
Fenotipo: Ojos oscuros

Minor allele frequency (MAF)

-the frequency at which the second most common allele occurs in a given population.

SNPs with a minor allele frequency of 5% or greater were targeted by the HapMap project.
It is widely used in population genetics studies because it provides information to differentiate
between common and rare variants in the population.

How to interpret MAF data

1. Introduce the reference of a SNP of interest, as an example: rs429358, in a database (dbSNP
or other).
2. Find MAF/MinorAlleleCount link. MAF/MinorAlleleCount: C=0.1506/754 (1000 Genomes)[3]
3. what do those numbers mean?:
a. C, is the minor allele for that particular locus
b. 0.1506, is the frequency of the C allele (MAF), it means 15% within the 1000 Genomes
database.
c. 754, is the number of times this SNP has been observed in the population of the study.

HERENCIA MITOCONDRIAL
Tradicionalmente se ha considerado que el ADN mitocondrial humano se hereda
slo por va materna.
Segn esta concepcin, cuando un espermatozoide fecunda un vulo penetra el
ncleo y su cola junto con sus mitocondrias son destruidos en el vulo materno.
Por lo tanto, en el desarrollo del cigoto slo intervendran las mitocondrias
contenidas en el vulo.
Sin embargo, se ha demostrado que las mitocondrias del espermatozoide pueden
ingresar al vulo. Segn algunos autores el ADN mitocondrial del padre puede
perdurar en algunos tejidos, como los msculos. Segn otros, no llega a heredarse
al ser marcado por ubiquitinacin y degradado.

Gene Structure

Most of the genes consist of short coding

sequences or exons, interrupted by a longer
intervening noncoding sequence or introns;
although a few genes in the human genome
have no introns.

DNA melting and annealing

Reversible
denaturation
and
annealing of
DNA

Melting point (tm) of DNA

The temperature at the mid-point of the transition
The tm of DNA depends on:
- Content of GC base pairs
- Size of DNA
- pH
- ionic strength

Restriction enzymes and genetic engineering

WE CAN HANDLE DNA BETTER THAN ANY OTHER BIOPOLYMER

Simple and universal chemistry
We can synthesize DNA: Make genes, manipulate organisms
We can sequence DNA: Genomics; great impact in Biomedicine and technology

We can modify NTs: DNA stability

We can modify DNA at our will: genetic engineering

Conclusions
DNA consists of four basesA, G, C, and Tthat are held in linear
array by phosphodiester bonds through the 3' and 5' positions of
adjacent deoxyribose moieties.
DNA is organized into two strands by the pairing of bases A to T
and G to C on complementary strands. These strands form a double
helix around a central axis.

The 3 x 109 base pairs of DNA in humans are organized into the
haploid complement of 23 chromosomes.
DNA provides a template for its own replication and thus
maintenance of the genotype and for the transcription of the
roughly 30,000 human genes into a variety of RNA molecules.

CURSO 0
LECTURE 2
DNA REPLICATION

THE CENTRAL DOGMA OF MOLECULAR BIOLOGY

The central dogma of molecular biology deals with the detailed residue-by-residue transfer
of sequential information. It states that such information cannot be transferred from
protein to either protein or nucleic acid
- Crick, Nature 227, 561 563 1970
The central dogma states that information in nucleic acid can be perpetuated or
transferred but the transfer of information into protein is irreversible. (B. Lewin, 2004)

DNA REPLICATION AND MUTATIONS

Cell Cycle

Mold

Copy

Cell division DNA replication (duplication)

Figure 6-6 Essential Cell Biology ( Garland Science 2010)

WHAT DO WE NEED FOR DNA REPLICATION

CHARACTERISTICS

RAPID: 10-1000 bp second

-So, how long does it take to synthesize the whole genome?
COMPLETE: synthesizes all bp
- What happens with telomers
ACCURATE: 1 error in 10 to the 5 bases
- Is this good enough?
- Proofreading

Studies in DNA replication led to molecular cloning, PCR, massive sequencing, and it
is the target of many anticancer or retroviral drugs.

THE CHEMISTRY OF DNA REPLICATION

Polarity: always synthesizes 5 to 3
Irreversible due to pyrophosphate release

THE REPLICATION FORK

Figure 6-12 Essential Cell Biology ( Garland Science 2010)

Figure 6-9 Essential Cell Biology ( Garland Science 2010)

THE ENZYME: DNA POLYMERASE

Arthur Kornberg 1956
won the nobel prize

SUBSTRATES NEEDED FOR THE

REACTION
PTJ: primer template junction
dNTPs: all 4 nucleotides triphosphate

STRUCTURE OF DNA POLYMERASE

Palm: binds PTJ and newly

synthesized strand. Binds the
backbone but also interacts with
base
Figure 6-14 Essential Cell Biology ( Garland Science 2010)

Fingers open and close

around the bound DNA
and incorporate the new
base

1 active site catalyzes addition of

multiple dNTPs because it works by
braille using the regular structure
of DNA

Its a matter of kinetics, the

addition of A is slower
because the P and OH are
not well aligned, so by the
time it takes it is most
probably washed away. The
addition of a T is favoured
because it is in the right
position

HOW DOES DNA POLYMERASE RECOGNIZE AND ATTACH TO ALL

DNA POSSIBLE CONFIGURATIONS

OTHER ENZYMES NEEDED FOR DNA REPLICATION

OTHER ENZYMES NEEDED FOR DNA REPLICATION

Topoisomerase

OTHER ENZYMES NEEDED FOR DNA REPLICATION

OTHER ENZYMES NEEDED FOR DNA REPLICATION

Helicase: unwinds DNA at the replication fork,
encircling just one strand of DNA. It is a
hexamer complex. It uses energy from ATP

With a rotational speed of up to 10,000 rotations per minute,

the helicase rivals the rotational speed of jet engine turbines.
Binding of a subunit to ATP causes the subunit to rotate 15 degree

TELOMERS: WHAT HAPPENS AT THE END OF THE CHROMOSOME

Repeat sequence of
TTAGGG in humans

Telomerase is a novel DNA

polymerase composed of
many proteins and a RNA
molecule (template)

Processive enzyme: repeats it 5060 times.

It only extends the 3 OH.
Sequence vary with different
organisms
It is regulated by the number of
repeats

Life as we know it (37C) exists thanks to the catalitic activity of enzymes

PCR

Preserving the information: repair of mutations

Mutations are the cause of inter-individual and inter-species variation and the cause
of diseases. Why do mutations occur?
A trait can be:
-Monogenic (around 4000
inherited diseases)
-Polygenic (predisposition,
inheritance + way of life)

Sickle cell anemia: genetic disease

which is caused by a single nucleotide
change in the 6th aa of the -chain of
hemoglobin (HBB gene)

Figure 6-19 Essential Cell Biology ( Garland Science 2010)

Alleles

LAS MUTACIONES

Table 6-1 Essential Cell Biology ( Garland Science 2010)

Genetic Variations
The genetic variations in DNA sequences (e.g.,
insertions, deletions, and mutations) have a
major impact on genetic diseases and
phenotypic differences.
All humans share 99% the same DNA sequence.
The genetic variations in the coding region may
change the codon of an amino acid and alters the
amino acid sequence.

Mutations

Caused by chance, genes, and environment

ultraviolet light
tobacco/alcohol
Can result in genetic coding of amino acids
Types
insertion
deletion
substitution
inversion (flipping)

Mutation classification:
- Synonymous: the substitution causes no
amino acid change to the protein it produces.
This is also called a silent mutation.
Non-Synonymous: the substitution results in
an alteration of the encoded amino acid. A
missense mutation changes the protein by
causing a change of codon. A nonsense
mutation results in a misplaced termination.

Mutations

Single Nucleotide Polymorphism

A Single Nucleotide Polymorphisms (SNP), pronounced
snip, is a genetic variation when a single nucleotide (i.e.,
A, T, C, or G) is altered and kept through heredity.
SNP: Single DNA base variation found >1%
Mutation: Single DNA base variation found <1%

94%

CTTAGCTT

99.9%

CTTAGCTT

6%

CTTAGTTT

0.1%

CTTAGTTT

SNP

Mutation

Mutations and SNPs

Observed genetic variations

SNPs

Mutations

Common
Ancestor

time

present

Single Nucleotide Polymorphism

SNPs are the most frequent form among various
genetic variations.
90% of human genetic variations come from SNPs.
SNPs occur about every 300~600 base pairs.
Millions of SNPs have been identified (e.g.,
HapMap and Perlegen).

SNPs have become the preferred markers for

association studies because of their high
abundance and high-throughput SNP genotyping
technologies.

Haplotypes
A haplotype stands for a set of linked SNPs on
the same chromosome.
A haplotype can be simply considered as a binary
string since each SNP is binary.
-A C T T T G C T C-

Haplotype 1

-A C T T A G C T T-

Haplotype 2

-A A T T T G C T C-

Haplotype 3

SNP1

SNP2

SNP3

SNP1

SNP2

SNP3

DNA mismatch repair is a system for recognizing and repairing erroneous insertion,
deletion, and mis-incorporation of bases that can arise during DNA
replication and recombination, as well as repairing some forms of DNA damage.

Enzymes involved in mismatch repair:

Causes colon cancer hereditary
It can pause/stop cell cycle if there
is a lot of damage
Leads to mutations in different
alleles if not repaired correctly

Base excision repair (BER) is a cellular mechanism that repairs damaged DNA throughout
the cell cycle. It is responsible primarily for removing small, non-helix-distorting base
lesions from the genome. BER is important for removing damaged bases that could
otherwise cause mutations by mispairing or lead to breaks in DNA during replication.
Common errors:
Add a ribonucleotide, roughly 1
every 10000 that has to be repaired

Oxydation of bases
Deamination or hydrolysis of bases
(for example C turns to U)

Methylations (for example

methylation of C, CpG islands)

Nucleotide excision repair (NER)

Nucleotide excision repair (NER) is a particularly important excision mechanism
that removes DNA damage induced by ultraviolet light (UV). The nucleotide excision
repair pathway repairs bulky helix-distorting lesions.