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2012 Univ. 1301 Aguilera Lecture
Introduction to Molecular and Cell Biology
Molecular biology seeks to understand
the physical and chemical basis of life.
and helps us answer the following?
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What is the molecular basis of disease?
What is the molecular basis of evolution ?
How did life arise on earth?
What is the molecular basis of memory?
How are different cell types produced from
a single embryo?
Other Important Questions?
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What are genes?
How do genes store information?
How is genetic information expressed?
How is this process regulated?
How are genes duplicated during cell
division?
• What are mutations?
To answer these questions it is necessary to
understand the nature of genes and proteins
Remembering what you already know
Prokaryotic Cell
Inner Membrane
Cell Wall
Outer Membrane
DNA
Eukaryotic Cell
DNA
Plasma Membrane
Nucleus
organelles
Nuclear Membrane
Prokaryotic Cells:
No nuclear membrane
No internal compartments
Eukaryotic Cells:
Nucleus
Specialized Organelles
Eukaryotic Membranes
Permeable to
gases and water
Active transport
requires energy
Genetic Information:
circular
E. coli
4 x 106 base pairs of DNA (1 chromosome)
max. potential proteins 3 x 103
actually 11-2 x 103
23 pairs
2.9 x 109 base pairs of DNA (46 chromosomes)
max. potential proteins 2.4 x 106
actually much less (~30(~30-40,000)
100--1000 times the total dry weight of bacteria
100
Human
cell
Human Chromosome pairs
Although human beings appear to be much more
complex and sophisticated than other
organisms, we contain and express very similar
genes that are highly evolutionarily conserved
The function of some genes is so important, that
they are highly conserved in sequence and
function
Expression of Genes follows an ordered developmental plan
HOX
Humans and Flies express similar genes but obviously have
different pattern of expression (function?)
During evolution, it is easier to adapt an existing gene than
to create it from scratch
Biological molecules are interdependent
Central Dogma
DNA
RNA
Protein
You need protein to make DNA/RNA
and you need nucleic acids to make proteins
• Prevalent view in early 1900’s was that genetic
information was contained within proteins
Why?
Proteins are more complex than nucleic acids (20
amino acids vs 4 different nucleotides)
Nucleic acids, DNA, was believed to play structural
role in cell
DNA is the Genetic Material
n
Early 1940’s DNA was finally implicated as the
genetic material
n
Pure DNA extracted from one bacterial strain
could provide genetic information to another
bacteria by a process known as transformation
n
Makes sense since DNA is very stable, it is
present in two copies in eukaryotes (diploid),
exist as doubledouble-stranded molecule, and
most importantly can be faithfully copied
Genes
Defined as a unit of DNA that encodes the
information for the synthesis of a protein
Prokaryotes contain less genetic information
(genes) than eukaryotes
Genes are copied (transcribed) into messenger
RNA and it is this message that is translated
into protein
In prokaryotes, genes involved in the same
pathway are commonly linked closed together
In most eukaryotes, each gene is generally
independently copied or transcribed into a single
RNA that is then translated into a single protein
Three genes in two chromosomes
Most genes in mammals and plants contain introns that are
removed during RNA processing
Introns spliced out (deleted)
mature message
Exons encode the information for protein synthesis
Mutations within genes can cause disease or death
Mutation in an exon can cause the production of an
abnormal or truncated (shorter) protein
Mutation in an intron can cause no effect or can
alter or destroy the normal processing of the mRNA
Mutation in regulatory regions can cause the gene to
not be expressed at all or over-expressed
Alternative Splicing can produce different or
altered proteins from the same gene
Fig.9-2
Even though humans encode for ~30,000 proteins
(based on gene count), the # of different proteins is
higher due to added complexity
In eukaryotes, each gene is independently
copied and generally encodes information for
a specific product (protein)
Eukaryotic mRNA is a product of several
modifications which include removal of introns
All DNA is arranged in a structure called a
double--helix that is composed of two identical
double
strands which adds to the chemical stability of
this molecule
Watson & Crick
DNA Double Helix
(1953)
Linkage of Nucleic Acids
Base 1
5’
O
Sugar OH HO P
Base 2
Sugar
O
O-H2O
5’
Base 1
Sugar
O
O
P
O-
Base 2
O Sugar
3’
3’
O-
O P O
Base 1
O
CH2 O
H
H
H
O H
O P O
O
H
Base 2
CH2 O
H H
H
3’
OH H
H
A
C
T
A
P
G
OH
5’ ACTAG 3’
UGAUC
T only in DNA
And U only in RNA
DNA Helix
Held by many
H bonds and
Hydrophobic
Interactions
Bases stack
on inside
Sugar Phosphate
backbone is on
the outside
To maintain
the geometry
of this structure
small bases,
pyrimidines,
(C or T) must
pair with larger
bases, purines,
(A or G)
A-T Base Pair
Thymidine (T)
CH3 C
Adenine (A)
O
H N H
C
C
N H
N
C N
CH
HC
C O
N
C
(Deoxyribose)
HC
N
C N
C
(Deoxyribose)
Base pair complementarity due to size, shape,
and chemical composition of bases
C-G Base Pair
Cytosine (C)
Guanine (G)
H N H
O
C
C
HC
N
HC
C O
N
C
(Deoxyribose)
H
H N
H
N
C N
CH
C
N
C N
C
(Deoxyribose)
Genes are arranged in Chromosomes
in specialized structures
Widely separated from each other
Humans and other higher organisms have more
junk DNA
DNA packaging within cells
The E. coli bacterial genome is approximately
1mm long which is about 1000 x the size (vol) of
a single bacteria
DNA needs to be highly compacted
Eukaryotic DNA
n
Can be up to hundred thousand times
longer than the cell that contains it
n
Found in highly compacted units called
chromosomes
n
DNA is wound around special proteins
Eukaryotic genes are arranged in chromosomes
gene 1
gene 2
gene 3
gene 4
Longest human chromosomes
2-3 x 108 base pairs ~10 cm long
Contents of Chromosomes
Chromosomes contain different types of sequences
such as:
n
Single copy genes, gene families, defective genes
n
Non-coding DNA and repetitive sequences
(can compose a significant part of genome)
n
Viral DNA and other transposable elements (few)
DNA cloning
Cloning comes from the greek word klon which means twig
Plants can be cloned by taking a cutting and replanting themthemthey should turn out identical to original plant.
The first cloning experiment was performed in 1973 (only 37
yrs ago) by Cohen and Boyer
They used bacterial plasmids which are small circular replicating
fragments of DNA
They also used enzymes that cut DNA into specific fragments.
These enzymes are called restriction endonucleases (enzymes
that cleave nucleic acids)
Plasmids are “selfish” pieces of DNA that are circular and replicate
inside of bacterial host
Origin of replication
Ampicillin resistance gene
When plasmids replicate they make two identical copies
Fig. 7.2
Restriction Enzyme Cleaves DNA at specific sites
They are called sticky ends because they have a tendency of pairing
or “re“re-pairing” with each other (or other foreign fragments)
Fragment of foreign DNA can be readily introduced into plasmids
Mix plasmid with E. coli bacteria and perform transformation
Heat Shock
Restriction Enzymes
Restriction enzymes (RE) are the bacteria’s defense against viruses
These enzymes restrict the host range of the viruses
There are more than 100 different restriction enzymes from a
large number of different bacteria
Biotech companies have been making millions of dollars selling
specific enzymes to researchers