From the Molecule to the

Cell

Introductory Lecture

Unity/Diversity
 4 masterpieces: a roman mosaic and 3 paintings

At first, we see a striking difference in styles: diversity

We realise subsequently that these are 4 portraits of human beings: unity

 From the molecule to the cell Introduction
Unity/Diversity

•The cell •Cloning and reverse genetics

•Gene expression
•Heredity
•Genomes
•Genes and enzymes
•Central dogma:
DNA, RNA, Proteins

7 chapters in biology have shaped our vision of biological

organisms: from micro-organisms to the human being.

Our perception of living organisms, from bacteria to human,

oscillates between unity and diversity
 Organism organ cell

The photoreceptor cell is a specialized type of neuron found in the retina that
is capable of phototransduction: the retinal cell converts light into electric
energy that will propagate from neuron to neuron.
The Cellular theory dates from 1838: “the cell is the
fundamental unit of life.”

The cellular theory is the first pillar of our vision of life.

Schleiden (1804-81) botanist: visualised cells when

microscopes were produced industrially (C. Zeiss).
He made a good observation but a wrong interpretation based on prior
ideology: the cell contains a “vital force” responsible for the organisation of
living organism, whereas current thinking is that cells and organisms follow
physics and chemistry laws.

Schwann (1810-82): physiologist and microscopist, studied

yeast, a microplant: described cells of the peripheral nervous
system which bear his name.
He also invented the term “metabolism”.
The Cellular theory (followed)

Virchow (1821-1902): Medical practitioner, founder of

human pathology.

More than a laboratory physician, Virchow was an impassioned

advocate for social and political reform. He stated that medicine is a
social science, and politics is nothing else but medicine on a large
scale.

Omnis cellula e cellula (1858)

Disease affects the organism but results from alterations of a cell (or a
group of cells).

Preventive medicine: public health, distribution of safe drinking water

and development of sewers for collection of waste water.
 The evolution theory is the second pillar of our vision of life.
Notion of common ancestor cell from which all living beings are derived.
The phylogenetic (evolutionary) tree of life

Notion of common ancestor cell

We visualise one tree: several details are not commonly accepted.
In the simple vision, the Darwinian one, mutations and selections are
sufficient, but does evolution occur quickly enough with these forces
only?

Yeast and man are very close on the evolutionary scale, and very far from E.Coli which
colonises our gut immediately after birth.
 The phylogenetic (evolutionary) tree of life: molecular scale

Nowadays, lines are based on DNA sequence (rRNA)

Ribosomal RNA is present in all cells, it probably appeared before DNA and was
probably present in the common ancestor.
The more the number of nucleotides differs, the further species are from each
other.
Archae which live mostly in extreme conditions were initially classified as bacteria.
We are aware now that they constitute a branch that is as distant from eukaryotes
as from bacteria (Carl Woese “discovered” the 3rd kingdom in 1977). There is recent
evidence of a common ancestor to archae and eukaryotes.
The cell membrane isolates two aqueous compartments

Lipids in our membranes are linear

molecules with two very different ends.
The head is polar (hydrophilic), the body
and the tail are non-polar and hydrophobic.

A double layer enables the membrane to

separate two aqueous compartments: inner
and outer environments.

There cannot be total integrity of the

membrane: there is a need for exchanges
between the cytoplasm and extracellular
space, as is provided by proteins.

Bi-layer:
a bi-dimensional fluid
 The cell membrane
Functions of membrane
in which proteins float
proteins:
•Channels (concentration
gradients).
•Pumps (energy requiring)
Transporters
Resistance (to
antibiotics,
chemotherapy)
•Recognition
•Markers of self
•Tissue adhesion

In E.Coli bacterium: 20% of genes encode membrane proteins

Theoretical representation of a human cell

Cells will be studied in Block Module B

 From the molecule to the cell: Introduction
Unity/Diversity

•The cell •Cloning and reverse genetics

•Gene expression
•Heredity
•Genomes
•Genes and enzymes
•Central dogma:
DNA, RNA, Proteins
 Mendel (1865): studied visible Laws of heredity
traits (phenotype) which reflect
influence of genes (genotype)
The theory of heredity is the 3rd pillar of
our vision of life.

Use of genetic knowledge goes back to Neolithic

period, with invention of agriculture and animal
breeding.

Mendel chose stable characters which always

showed in the first generation during cross
breeding phenotypes which were identical to one
of the two parents and not an intermediate
phenotype between the two parents.

The study of 7 characters enables us to

generalise the model.
Here are 3 of the 7 characters that were studied by Gregor Mendel: 3 traits above are
dominant, flower position is a recessive trait
 Genetics: incorporates a combination of 2 opposed
characteristics, constancy and variation
Wild type: is the reference organism, which is arbitrarily
chosen!
Mutant: variant (rare) which has a stable phenotype over
time, *generated by a change in DNA sequence*.
Epigenetic change: covalent (stable) modification of DNA or
of chromatin which does not change the sequence, but
modifies gene expression.
An epigenetic variant is less stable, in successive cell divisions, than a mutant.

Most of the offspring are identical to their parents if both are

homozygotes, this is genetic constancy.
Rare individuals seem different, and the difference appears in the
offspring, in proportions according to Mendel’s laws.
Mutants occur spontaneously (DNA replication errors or accidents) or
may be induced by mutagenic agents (X rays, UV rays, chemical products)
 D. Melanogaster
And T.H Morgan school of
thought

Chromosomal
theory of heredity

A. Sturtevant (1912) Dozens of mutants are identified and cross-bred.

•If 2 mutations are on different chromosomes, they segregate independently (2nd
Mendel’s law of independent assortment)
•If the 2 mutations are on the same chromosome, we observe a “linkage” of
mutations, more or less important depending on the physical distance between
them.
This type of analysis enables genetic mapping where each locus is placed on the
chromosome.
 Chromosomal map in Medicine

Cri du Chat Syndrome

Partial deletion of chromosome 5

Deletions and rearrangements are Phenotype: Malformations and

mental retardation.
observed in many cancers. We talk about haploinsufficiency here
Boveri (1902): observed a link because a single copy of the Ch 5
region is not sufficient for normal
between aneuploidy and cancer. development.
 From the molecule to the cell: Introduction
Unity/Diversity

•The cell •Cloning and reverse genetics

•Gene expression
•Heredity
•Genomes
•Genes and enzymes
•Central dogma:
DNA, RNA, Proteins
 Hypothesis: One gene= one enzyme
•Alkaptonuria:
Urine that blackens babies’ nappies.
One enzyme is missing in patients.
First “mutant” humans.
1st connection between genes and enzymes by
(Garrod, in 1902).

For Mendel and Morgan, genes are abstract notions without

a definite physical support. There was a void between
genotype and phenotype!

Garrod observed that alkaptonuric babies were born from

cousins (where both parents were heterozygous).
Parents were normal as the mutation is recessive and one
allele produces enough enzymes.
 Study of pigment in Fly’s eye: Ephrussi and Beadle (1935)

Many genes are needed to

synthesise the red pigment:
notion of metabolic pathway.

Wild type One of the mutants

Different fly mutants have different eye colours.

These studies helped to isolate intermediates and the end

product of red pigment.

The choice of study organism is important for improving on

knowledge.
 Beadle and Tatum (1941)
Studied Fungi (Neurospora crassa) and bacteria (E. Coli): they could grow on a
simple medium (salts and glucose) or in a rich medium.
•Wild type (prototroph): grows on a simple medium.
•Mutants (auxotrophs): do not grow on a simple medium, they grow on a
rich medium.
Most of the mutants could not synthesise: one amino acid, one
nucleotide, or one vitamin. There was a defect in one step, catalysed by
one enzyme.
“genes act by regulating definite chemical events”

The choice of organism is important to improve on our

knowledge. Bacteria offer another advantage: they are haploid.
Micro-organisms grow on different media. If they need only glucose as a source of
carbon, they are capable of synthesising amino acids, vitamins, nucleotides, all the
small molecules that are necessary for life.
A simple medium contains glucose as sole organic molecule, ammonium (nitrogen
source) and salts (phosphates, sulfates, iron, magnesium, calcium).
A rich medium additionally contains amino acids, vitamins and nucleotides.
Schematic representation
of intermediate
metabolism looks like this
nowadays
 One segment in the map of intermediate metabolism (1/115)
Transformation of small molecules

The main enzyme in this segment is β-galactosidase.

The enzyme converts lactose (substrate) into glucose + galactose (end product). These
reactions require energy (they are energy dependent), the main cellular source of
energy is ATP.
Oxidations (loss of electrons) and reductions (gain of electrons) occur with electron
acceptors such as NAD+ or electron donors such as NADH
 Cells obey to chemistry laws

Enzymes speed up
chemical reactions without
modifying their
equilibrium.

The majority of enzymes

are proteins, but not all of
them!

Enzymes= catalysts
 Enzymes can also be
RNA: ribozymes

RNA at the origin of life:

replication and catalysis

Replication and coding activities of some RNA are well known (flu virus,
mRNA…).
The ability of some RNA to function as enzymes is a novel discovery.
Many small RNAs participate to control of gene expression.
They are used in the laboratory to selectively modulate expression of genes.
Two experimental approaches are used , each
having its own limitations

Biochemistry: a reaction may occur but may have no

physiological importance

Genetics: A gene product may participate in a pathway

in a very indirect manner
 A biochemical reaction may occur
On this pancreatic section we
see an islet of Langerhans (in
violet, area where pro-insulin is
synthesised) and the exocrine
portion (pink, where digestive
enzymes are synthesised
including trypsin).
Trypsin may convert proinsulin
into insulin Trypsin and pro-insulin never
come in contact in normal
conditions, the former is
secreted in the gut, and the
latter goes directly into blood.
 From the molecule to the cell: Introduction
Unity/Diversity

•The cell •Cloning and reverse genetics

•Gene expression
•Heredity
•Genomes
•Genes and enzymes
•Central dogma:
DNA, RNA, Proteins
 The central dogma in molecular biology (Crick, 1958)

DNA RNA Protein

•All the polymers are linear
•2 alphabets: 4 bases and 20 amino acids (standard sets)
•All the information lies in the sequence
•Transmission:
a)Base-pairing
b)Genetic code
The double helix structure of DNA proposed by Watson and Crick in 1953 offers
an ideal model for replication.
Information transmission DNA-DNA and DNA-RNA implies base-pairing
according to Watson-Crick rules : A and T, G and C
The Genetic code specifies which amino acid corresponds to which base triplet.
The proposed model helped to solve the problem of 3-D structures. They are
determined only by sequence
Central dogma revisited, Crick in 1970
What types of information transfer occurs
between the macromolecules?

General (probable)

Special (possible)

Non-detected (impossible)

The absent arrows are important!! 1970 1958

Special reactions that are frequently encountered: replication of RNA virus (flu
virus….), DNA synthesis using genomic RNA of retrovirus (HIV…).
Protein synthesis from DNA has been observed in the laboratory only.
Reactions described as impossible in 1958 have never been detected to date.
 From the molecule to the cell: Introduction
Unity/Diversity

•The cell •Cloning and reverse genetics

•Gene expression
•Heredity
•Genomes
•Genes and enzymes
•Central dogma:
DNA, RNA, Proteins
CLONING (Immortalisation of one DNA molecule)

Use of plasmids (mini-chromosomes), cleavage by restriction enzymes and

then combining the plasmid DNA + DNA fragment to be cloned by DNA
ligase.
Then recombinant DNA is introduced into a bacterium, those with the
recombinant DNA are selected, and allowed to proliferate by cell division
(and are immortalised).
A human gene can function in a bacterium, the enzyme it encodes can
replace the corresponding bacterial enzyme.
DNA can be copied from mRNA by using a reverse transcriptase, and then
cloned (copy of the mRNA is cloned) .
 CLONING AND GENETIC ENGINEERING

Recombinant proteins play an essential role in

medicine.
•Thrombolytic treatment in myocardial infarction.
•Insulin treatment in Diabetes .
•Interferon in Multiple Sclerosis (MS) (Sclérose en plaque or
SEP in French).

•Immunisation.
•Chemotherapy.
 REVERSE GENETICS
•Classical genetics: from the phenotype of a mutant to the
gene.
•Genetic engineering enables reverse genetic studies:
From a gene which has been specifically mutated
to a phenotype.

•Genes can be knocked in or

knocked out in the mouse.
•Useful to reproduce and to
better understand a
pathology and to develop a
therapy.

We will study an example of classical genetics with the Case Study on Cystic
Fibrosis.
 From the molecule to the cell: Introduction
Unity/Diversity

•The cell •Cloning and reverse genetics

•Gene expression
•Heredity
•Genomes
•Genes and enzymes
•Central dogma:
DNA, RNA, Proteins
 GENE EXPRESSION
Gene expression: we need to answer 3 questions for a gene,
where, how much and when?
Albumin Factor IX IgG Globins*
Site plasma Red blood
cells
Synthesis Hepatocytes lymphocytes reticulocytes
Quantity +++++ + ++/++++ +++++
* Type of globin varies (embryo, foetus, adult)

Most cells contain all genetic information.

All genes are not expressed in the same way: “Housekeeping” genes are
expressed in most of the cells, specialised genes are expressed only in some
cellular types.
Some genes are variably expressed depending on different conditions such
as: development, normal physiology, pathologic conditions or in different
phases of the cell cycle.
 GENE EXPRESSION The lactose operon (Jacob & Monod, 1960)

Bacterium E.Coli often used to study gene expression:

DNA is constituted by 3 types of elements:
ZYA: genes encoding structural proteins and enzymes.
I: Regulator genes (repressors and activators)
O: DNA sites (elements) interacting with regulating proteins

Without lactose With lactose

I P O Z Y A I P O Z Y A

Transcription No Transcription Transcription

transcription
mRNA mRNA

Translation
No mRNA
Translation
X mRNA

Translation
NO EXPRESSION Repressor
Repressor
(tetramer) EXPRESSION
Inductor (lactose or IPTG)
Initial observation was simple:
•Without lactose the wild type cell did not contain an enzyme
that was capable of degrading lactose into glucose + galactose.
•When lactose was added to the medium, the enzyme was
synthesised.
This is an example of negative control, we are aware now of
many examples of genes whose expression is positively
controlled.
Lactose is both an inductor and a substrate.
Inductor: prevents binding of the repressor to the operator site.
Substrate: lactose is hydrolysed by the Z gene product into
galactose + glucose.
Enzyme encoded by gene Z is β-galactosidase.
Mutant bacteria were described that constitutively expressed Z
gene: either because of mutant I gene (produced an inactive
repressor) or because site O was mutated and inactive.
Lactose operon: there are different mutations inducing constitutive synthesis
of β-galactosidase.
1-Inactive repressors such as mutated i gene (recessive) which regulates
gene encoding β-galactosidase.
2-Inactive operator site (dominant) such as a mutated O site.
In both cases, genes are expressed at all times, even in the absence of
lactose.

What will happen if an intact i gene regulator, carried in a plasmid for

example, is introduced in a cell with an inactive i? Its product, the
repressor, will prevent the synthesis of enzymes in the absence of lactose.
We will find again the wild type phenotype (inducible). The mutant is
recessive.

What will happen if we introduce a whole intact operon on a plasmid? The

wild type operon in the plasmid is expressed only in presence of lactose
(inducible). The operon containing the inactive operon site is expressed all
the time (constitutive). The mutant is dominant.
 Bacteriophages: pathogens

The wild type λ virus particle (virion) contains a genome close to

50 kb of linear double-stranded DNA packaged within a protein
coat and has evolved a highly efficient mechanism of infecting E.
Coli cells.

After the λ virion has attached to the bacterial wall, the coat
protein is discarded and the λ DNA is injected into the cell.

The λ DNA can enter two alternative pathways: lytic cycle or the
lysogenic state.

In the lytic cycle: λ DNA replicates, coat proteins are

synthesised, some of the λ gene products lyse the cell, allowing
the virion to escape and infect new cells.
 In the lysogenic state: the λ
genome possesses a gene att which
has a homolog in the E.Coli
chromosome.
Apposition of the two att genes can
result in recombination between
the λ and the E.Coli genomes and
subsequent integration of the λ
DNA within the E.Coli chromosome.
In this state the λ DNA is described
as a provirus and the host cell a
lysogen because the λ DNA can
remain stable for long periods but
has the capacity for excision from
the host chromosome and entry
Lysogenic into a lytic cycle.
Lytic cycle Ly
pathway
Lysogen: a repressor that prevents
Immunity: a lysogen that prevents
expression of viral structural genes
cell lysis by a 2nd bacteriophage λ
and cell lysis.

A lysogenic bacterium does not produce viruses, it can be induced, by UV for

example, to enter into a lytic cycle.
A lysogenic bacterium can be infected a second time by the same virus. In
that case, the second virus is blocked by a repressor induced by the first one.
The bacterium is said to be immune to the virus.

Viruses that are incapable of lysogenicity are different from wild type viruses. Some
have an inactive repressor, others have an inactive operator site. The two types can be
distinguished by infection of a bacterium which is lysogenic for the wild type.
 From the molecule to the cell: Introduction
Unity/Diversity

•The cell •Cloning and reverse genetics

•Gene expression
•Heredity
•Genomes
•Genes and enzymes
•Central dogma:
DNA, RNA, Proteins
 Genomes: the molecular anatomy

1976: MS2 (bacterial RNA virus) 3, 569 bases

1978: ΩX174 (bacterial DNA virus) 5, 386 bases

1995: Haemophilus influenzae 1, 830,137 bases

1997: Saccharomyces cerevisiae 13, 117, 000 bases
2000: Homo Sapiens -3 200, 000, 000 bases
2002: Mouse
2004: Rat
2007: 353 bacteria and 38 archae
 Mycobacterium tuberculosis: 3924
Escherichia coli genes
Mycobacterium leprae: 1604 genes
Mycoplasma: 477 genes
Origin of
Replication Carsonella” 185 genes
E. Coli : 4286 genes

Terminus of
replication

Yeast has around 6000 genes, fly have some 14 000 genes, and
humans have around 25 000 genes.
 Vertical evolution of species

Horizontal transfer of genes between

species: accelerated evolution

Virus The best known and massive

Plasmids transfer: acquisition of bacterium
Naked DNA which is ancestor of mitochondria,
and of another bacterium that is an
ancestor of chloroplasts.
Bacteria often become pathogenic by acquiring plasmids or lysogenic bacteria:
• Pathogenic strains of Anthrax bacillus contain two plasmids that encode toxins.
• The most pathogenic strains of Klebsiella have incorporated up to 18 genomes of
lysogenic phages.
Viruses Stable latent “free “organisms, obligatory
parasites. There are > 2000 viral genomes

Polio virus
Phage T4
Measles
 Bacteriophages: pathogens
Cholera, diphteria: toxins secreted by these bacteria are encoded
by genes of lysogenic bacteriophages similar to λ.
Some of these toxins help to synthesise very effective vaccines
(diphteria) but not all of them.

Bacteriophages: A forgotten treatment

Treatment of an
infection by
bacteriophages
 Viruses: A great reservoir of genes
300 genes:
60 essential genes
120 non essential genes (known enzymes)
120 genes with unknown function

Phages: 10
millions/ml of
sea water,
Total mass?

“Unknown” genes are coding sequences often

called ORFans (Open Reading Frame) by analogy
to orphans, they have no defined function yet
and no homologs in genomes sequenced to
Pandoravirus: 2600 “genes”. date.
Only 400 have known homologs, They represent the “dark matter” in the tree of
>85% ORFans. life….