Introduction to Genome

Biology

Sandrine Dudoit and Robert Gentleman

Bioconductor Short Course

2003

© Copyright 2002, all rights reserved

 Outline
• Cells, chromosomes, and cell division
• DNA structure and replication
• Proteins
• Central dogma: transcription, translation
• Microarrays
• Pathways
A brief history
Gregor Mendel (1823-1884)

Thomas Hunt Morgan (1866-1945)

Francis Crick (1916- )

James D. Watson (1928- )

From chromosomes to
proteins
Cells
 Cells

• Cells: the fundamental working

units of every living organism.

• Metazoa: multicellular organisms.

E.g. humans: trillions of cells.
• Protozoa: unicellular organisms.
E.g. yeast, bacteria.
 Cells
• Each cell contains a complete copy of
an organism’s genome, or blueprint for
all cellular structures and activities.

• Cells are of many different types (e.g.

blood, skin, nerve cells), but all can be
traced back to a single cell, the fertilized
egg.
 Cell composition
• 90% water.
• Of the remaining molecules, dry weight
– 50% protein
– 15% carbohydrate
– 15% nucleic acid
– 10% lipid
– 10% miscellaneous.
• By element: 60% H, 25% O, 12%C, 5%N.
 The genome
• The genome is distributed along
chromosomes, which are made of
compressed and entwined DNA.

• A (protein-coding) gene is a
segment of chromosomal DNA that
directs the synthesis of a protein.
 The human genome
• The human genome is distributed
along 23 pairs of chromosomes
–22 autosomal pairs;
–the sex chromosome pair, XX for
females and XY for males.
• In each pair, one chromosome is
paternally inherited, the other
maternally inherited (cf. meiosis).
Chromosomes
Chromosome banding
patterns
Of mice and men
Chromosomes and DNA
 DNA structure
“We wish to suggest a
structure for the salt of
deoxyribose nucleic acid
(D.N.A.). This structure
has novel features which
are of considerable
biological interest.”

J.D. Watson & F. H. C. Crick. (1953). Molecular structure of Nucleic Acids. Nature. 171: 737-738.
 DNA structure
• A deoxyribonucleic acid or DNA molecule is
a double-stranded polymer composed of four
basic molecular units called nucleotides.
• Each nucleotide comprises
– a phosphate group;
– a deoxyribose sugar;
– one of four nitrogen bases:
• purines: adenine (A) and guanine (G),
• pyrimidines: cytosine (C) and thymine (T).
DNA structure
DNA structure
 Nucleotide bases
Purines

Adenine (A) Guanine (G)

Pyrimidines

Thymine (T) Uracil (U)

Cytosine (C)
(DNA) (RNA)
 DNA structure
• Polynucleotide chains are directional
molecules, with slightly different structures
marking the two ends of the chains, the so-
called 3' end and 5' end.
• The 3' and 5' notation refers to the numbering
of carbon atoms in the sugar ring.
• The 3' end carries a sugar group and the 5'
end carries a phosphate group.
• The two complementary strands of DNA are
antiparallel (i.e, 5' end to 3' end directions for
each strand are opposite)
 The human genome in
numbers

• 23 pairs of chromosomes;
• 2 meters of DNA;
• 3,000,000,000 bp;
• 35 M (males 27M, females 44M);
• 30,000-40,000 genes.
 DNA replication

“It has not escaped our

notice that the specific
pairing we have
postulated immediately
suggests a possible
copying mechanism for
the genetic material.”

J.D. Watson & F. H. C. Crick. (1953). Molecular structure of Nucleic Acids. Nature. 171: 737-738.
DNA replication

Three possible
models
DNA replication
 DNA replication

• In the replication of a double-stranded or duplex

DNA molecule, both parental (i.e. original) DNA
strands are copied.
• The parental DNA strand that is copied to form a
new strand is called a template.
• When copying is finished, the two new duplexes
each consist of one of the original strands plus its
complementary copy - semiconservative
replication.
DNA replication

Base pairing provides

the mechanism for
DNA replication.
 DNA replication
• Many enzymes are required to unwind
the double helix and to synthesize a
new strand of DNA.
• The unwound helix, with each strand
being synthesized into a new double
helix, is called the replication fork.
• DNA synthesis occurs in the 5' Æ 3'
direction.
DNA replication
DNA replication
Enzymes in DNA replication
1. Topoisomerase: removes supercoils and initiates
duplex unwinding.
2. Helicase: unwinds duplex.
3. DNA polymerase: synthesizes the new DNA
strand; also performs proofreading.
4. Primase: attaches small RNA primer to single-
stranded DNA to act as a substitute 3'OH for DNA
polymerase to begin synthesizing from.
5. Ligase: catalyzes the formation of phosphodiester
bonds.
6. Single-stranded binding proteins: maintain the
stability of the replication fork.
Proteins
 Proteins
• Proteins: large molecules composed of one
or more chains of amino acids, polypeptides.
• Amino acids: class of 20 different organic
compounds containing a basic amino group
(-NH2) and an acidic carboxyl group (-
COOH).
• The order of the amino acids is determined
by the base sequence of nucleotides in the
gene coding for the protein.
• E.g. hormones, enzymes, antibodies.
Amino acids
Amino
acids
Amino acids
Proteins
Proteins
Cell types
 Differential expression
• Each cell contains a complete copy of the
organism's genome.
• Cells are of many different types and states
E.g. blood, nerve, and skin cells, dividing cells,
cancerous cells, etc.
• What makes the cells different?
• Differential gene expression, i.e., when,
where, and how much each gene is
expressed.
• On average, 40% of our genes are expressed
at any given time.
Central dogma
 Central dogma
The expression of the genetic information
stored in the DNA molecule occurs in two
stages:
– (i) transcription, during which DNA is
transcribed into mRNA;
– (ii) translation, during which mRNA is
translated to produce a protein.
DNA Î mRNA Î protein
Other important aspects of regulation:
methylation, alternative splicing, etc.
Central dogma
 RNA
• A ribonucleic acid or RNA molecule is a
nucleic acid similar to DNA, but
– single-stranded;
– ribose sugar rather than deoxyribose sugar;
– uracil (U) replaces thymine (T) as one of the
bases.
• RNA plays an important role in protein
synthesis and other chemical activities of the
cell.
• Several classes of RNA molecules, including
messenger RNA (mRNA), transfer RNA
(tRNA), ribosomal RNA (rRNA), and other
small RNAs.
 The genetic code
• DNA: sequence of four different nucleotides.
• Proteins: sequence of twenty different
amino acids.
• The correspondence between DNA's four-
letter alphabet and a protein's twenty-letter
alphabet is specified by the genetic code,
which relates nucleotide triplets or codons to
amino acids.
The genetic code
Start codon: initiation of
translation (AUG, Met).
Stop codons: termination
of translation.

Mapping between codons and

amino acids is many-to-one:
64 codons but only 20 a.a..

Third base in codon is often

redundant,
e.g., stop codons.
Protein synthesis
 Transcription
• Analogous to DNA replication: several steps and
many enzymes.

• RNA polymerase synthesizes an RNA strand

complementary to one of the two DNA strands.

• The RNA polymerase recruits rNTPs (ribonucleotide

triphosphate) in the same way that DNA polymerase
recruits dNTPs (deoxunucleotide triphospate).

• However, synthesis is single stranded and only

proceeds in the 5' to 3' direction of mRNA (no
Okazaki fragments).
 Transcription
• The strand being transcribed is called the
template or antisense strand; it contains
anticodons.

• The other strand is called the sense or

coding strand; it contains codons.

• The RNA strand newly synthesized from and

complementary to the template contains the
same information as the coding strand.
Transcription
 Transcription
• Promoter. Unidirectional sequence upstream
of the coding region (i.e., at 5' end on sense
strand) that tells the RNA polymerase both
where to start and on which strand to
continue synthesis. E.g. TATA box.
• Terminator. Regulatory DNA region
signaling end of transcription, at 3' end .
• Transcription factor. A protein needed to
initiate the transcription of a gene, binds
either to specific DNA sequences (e.g.
promoters) or to other transcription factors.
Transcription
 Exons and introns
• Genes comprise only about 2% of the human
genome.
• The rest consists of non-coding regions
– chromosomal structural integrity,
– cell division (e.g. centromere)
– regulatory regions: regulating when, where, and in
what quantity proteins are made .
• The terms exon and intron refer to coding
(translated into a protein) and non-coding
DNA, respectively.
Exons and introns
Splicing
 Translation
• Ribosome:
– cellular factory responsible for protein synthesis;
– a large subunit and a small subunit;
– structural RNA and about 80 different proteins.
• transfer RNA (tRNA):
– adaptor molecule, between mRNA and protein;
– specific anticodon and acceptor site;
– specific charger protein, can only bind to that
particular tRNA and attach the correct amino acid
to the acceptor site.
 Translation
• Initiation
– Start codon AUG, which codes for methionine,
Met.
– Not every protein necessarily starts with
methionine. Often this first amino acid will be
removed in post-translational processing of the
protein.
• Termination:
– stop codon (UAA, UAG, UGA) ,
– ribosome breaks into its large and small subunits,
releasing the new protein and the mRNA.
Translation
tRNA
• The tRNA has an
anticodon on its
mRNA-binding end that
is complementary to the
codon on the mRNA.
• Each tRNA only binds
the appropriate amino
acid for its anticodon.
 Alternative splicing
• There are more than 1,000,000 different
human antibodies. How is this possible with
only ~30,000 genes?
• Alternative splicing refers to the different
ways of combining a gene’s exons. This can
produce different forms of a protein for the
same gene.
• Alternative pre-mRNA splicing is an important
mechanism for regulating gene expression in
higher eukaryotes.
• E.g. in humans, it is estimated that
approximately 30% of the genes are subject
to alternative splicing.
Alternative splicing
 Immunoglobulin
• B cells produce antibody
molecules called immunoglobulins
(Ig) which fall in five broad
classes.

• Diversity of Ig molecules
– DNA sequence: recombination,
mutation.
– mRNA sequence: alternative
splicing.
– Protein structure: post-translational IgG1
proteolysis, glycosylation.
Post-translational processing
• Folding.
• Cleavage by a proteolytic (protein-cutting)
enzyme.
• Alteration of amino acid residues
– phosphorylation, e.g. of a tyrosine residue.
– glycosylation, carbohydrates covalently attached
to asparagine residue.
– methylation, e.g. of arginine.
• Lipid conjugation.
 Transcription and translation
transcription

DNA translation

mRNA

Protein

Regulatory
Organism network
 Control of Gene Expression
• there is strong evidence that the DNA
content of most cells in a multi-cell
organism is identical
• different cell types synthesize different
sets of proteins at different times
Gene expression
 Control of Gene Expression
• there are at least six ways to control
protein expression
1. control when and how often a gene is
transcribed
2. control how the transcript is spliced
3. select which mRNA's are exported from
the nucleus
4. control translation
 Controlling Expression
5. selectively destabilize mRNAs in the
cytoplasm
6. control protein activity (degradation,
inactivate, isolate), post-translational
modifications
• for most genes transcriptional control
is the most important
 Gene Expression
• for many diseases specific patterns of
gene expression (mRNA expression)
have been associated with the different
phenotypes
 Class
Class discovery
discovery

Different Tumors have

different patterns of Fig. from Pomeroy et al. Nature 415 (2002)

expression
 DNA Microarrays
• the data obtained from microarray
experiments is a measure of the
abundance of a nucleic acid
• usually they are used for detecting
mRNA levels
• some of the issues mentioned
previously can affect the observed
abundance of mRNA
 Low values of mRNA
• the gene may be deleted
• the gene may be being repressed
• the gene may no longer be enhanced
• the gene may be methylated
• the mRNA may be kept in the nucleus
 High Levels of mRNA
• the gene may be part of an amplicon
• the gene may no longer be being
repressed
• the gene may be being enhanced
 Other Issues
• was the right sequence applied to the
chip?
• alternative splicing: which one are we
measuring?
• cross-hybridization – genes with similar
sequences may hybridize
 An example of the interactions between
some genes (adapted from Wagner 2001)

protein
phosphatase transcription
factor
transcription inactive inactive
protein
factor P
kinase

protein
P
active active

DNA Gene 1 Gene 2 Gene 3 Gene 4 Gene 5

 Downstream Consequences
• many genes fall into the class of genes
called transcription factors
• while most genes are transcribed by
RNA polymerase II it cannot initiate
transcription itself in eukaryotic cells
• transcription factors identify and then
bind to specific sites in the DNA
• the TFs then guide and activate RNA
polymerase
 Downstream Consequences
• TFs tend not to be specific for one gene
• disregulation (or over or under
production) of a TF can have large
effects on gene expression
• for example ESR1 (estrogen receptor 1)
is a transcription factor
• it affects production of cyclin d1
(CCND1)
 Downstream Consequences
• CCND1 forms a complex with CDK4
and/or CDK6
• this complex inactivates the repressor
function of pRb (retinoblastoma protein)
which regulates cell proliferation
• and so on….
 Functional genomics
• The various genome projects have yielded
the complete DNA sequences of many
organisms.
E.g. human, mouse, yeast, fruitfly, etc.
Human: 3 billion base-pairs, 30-40 thousand genes.
• Challenge: go from sequence to function,
i.e., define the role of each gene and
understand how the genome functions as a
whole.
 Pathways
• The complete genome sequence doesn’t tell
us much about how the organism functions
as a biological system.
• We need to study how different gene
products interact to produce various
components.
• Most important activities are not the result of
a single molecule but depend on the
coordinated effects of multiple molecules.
 TGF-β pathway
• Transforming Growth Factor beta, TGF-β,
plays an essential role in the control of
development and morphogenesis in
multicellular organisms.

• The basic pathway provides a simple route

for signals to pass from the extracellular
environment to the nucleus, involving only
four types of molecules.
TGF-β pathway

Four types of molecules

• TGF-β
• TGF-β type I receptors
• TGF-β type II receptors
• SMADS, a family of signal
transducers and
transcriptional activators.
TGF-β pathway
 TGF-β pathway
• Extracellular TGF−β ligands transmit
their signals to the cell's interior by
binding to type II receptors, which form
heterodimers with type I receptors.

• The receptors in turn activate the SMAD

transcription factors.
 TGF-β pathway
• Phosphorylated and receptor-activated
SMADs (R-SMADs) form heterodimers
with common SMADs (co-SMADs) and
translocate to the nucleus.

• In the nucleus, SMADs activate or

inhibit the transcription of target genes,
in collaboration with other factors.
 Pathways
• [Link]
• There are many open questions
regarding the relationship between gene
expression levels (e.g. mRNA levels)
and pathways.
• It is not clear to what extent microarray
gene expression data will be
informative.
 WWW resources
• Access Excellence
[Link]
• Genes VII
[Link]
• Human Genome Project Education Resources
[Link]
• Kimball’s Biology Pages
[Link]
• MIT Biology Hypertextbook
[Link]