4
Fundamentals of
Molecular Biology
4 Fundamentals of Molecular Biology
•
Heredity, Genes, and DNA
•
Expression of Genetic Information
•
Recombinant DNA
•
Detection of Nucleic Acids and
Proteins
•
Gene Function in Eukaryotes
Introduction
Molecular biology focuses on the
mechanisms of transmission and
expression of genetic information.
Fundamental experiments on model
organisms such as bacteria and viruses
have provided information on molecular
mechanisms that operate in all
organisms.
Introduction
Recombinant DNA technology allowed
fundamental principles and experimental
approaches to be extended to eukaryotic
cells.
Recombinant DNA allows isolation and
characterization of individual genes, and
determination of complete genome
sequences.
Heredity, Genes, and DNA
All organisms inherit genetic information
specifying their structure and function
from their parents.
All cells arise from preexisting cells, so
the genetic material must be replicated
and passed from parent to progeny at
each cell division.
Heredity, Genes, and DNA
The classical principles of genetics were
deduced by Gregor Mendel in 1865,
from experiments with pea plants.
He studied well-defined traits such as
seed color, and could predict patterns
of inheritance by assuming that each
trait is determined by a pair of inherited
factors, now called genes.
Heredity, Genes, and DNA
One gene copy (allele) specifying each
trait is inherited from each parent.
Example: Plants with identical alleles
specifying yellow (YY) or green (yy)
seeds, are crossed.
The progeny (F1 generation) are hybrids
(Yy) and have yellow seeds: yellow is
dominant, green is recessive.
Figure 4.1 Inheritance of dominant and recessive genes
Heredity, Genes, and DNA
Genotype is the genetic makeup of an
individual.
Phenotype is the resulting physical
appearance.
The genotype of the F1 generation is Yy.
The phenotype is yellow.
Heredity, Genes, and DNA
Mendel’s results were ignored until 1900
when their importance was recognized.
Shortly afterward, chromosomes were
identified as the carriers of genes.
Heredity, Genes, and DNA
Most cells of plants and animals are
diploid: they have two copies of each
chromosome.
Formation of germ cells (sperm and egg)
involves a type of cell division
(meiosis) in which only one member of
each chromosome pair is transmitted to
each progeny cell.
Figure 4.2 Chromosomes at meiosis and fertilization
Heredity, Genes, and DNA
The sperm and egg are haploid: they
have only one copy of each
chromosome.
At fertilization, two haploid cells are
combined to make a new diploid cell.
Heredity, Genes, and DNA
Fundamentals of mutation, genetic
linkage, genes, and chromosomes
were established by experiments with
the fruit fly Drosophila melanogaster.
In the early 1900s, mutations (genetic
alterations) were observed in
Drosophila that affected characters
such as eye color and wing shape.
Heredity, Genes, and DNA
Breeding experiments showed that some
genes are inherited independently of
each other.
This suggested that the genes are on
different chromosomes that segregate
independently during meiosis.
Genes located on the same chromosome
are inherited together and are said to be
linked.
Figure 4.3 Gene segregation and linkage (Part 1)
Figure 4.3 Gene segregation and linkage (Part 2)
Figure 4.3 Gene segregation and linkage (Part 3)
Figure 4.3 Gene segregation and linkage (Part 4)
Heredity, Genes, and DNA
The relationship between genes and
proteins began to emerge in 1909:
• It was realized that the inherited
disease phenylketonuria results
from a genetic defect in the enzyme
needed to metabolize the amino
acid phenylalanine.
Heredity, Genes, and DNA
In 1941, experiments with the fungus
Neurospora crassa by Beadle and
Tatum found more evidence linking
genes with the synthesis of enzymes.
Mutant strains of Neurospora required
particular amino acids for growth.
Heredity, Genes, and DNA
Each mutation resulted in a deficiency in
a specific metabolic pathway, which is
governed by enzymes.
This led to the conclusion that genes
specify enzyme structure.
It is now known that some genes specify
RNAs rather than proteins.
Heredity, Genes, and DNA
Evidence that DNA is the genetic
material first came from experiments
with the bacterium that causes
pneumonia (Pneumococcus).
A “transforming principle” was
responsible for inducing the genetic
transformation of one strain of the
bacteria to another.
Figure 4.4 Transfer of genetic information by DNA
Heredity, Genes, and DNA
The transforming principle was later
identified as DNA when it was shown
that the activity of the transforming
principle is abolished by enzymatic
digestion of DNA, but not by digestion
of proteins.
Heredity, Genes, and DNA
Other studies with viruses confirmed that
DNA was the genetic material.
It was shown that when a bacterial virus
infects a cell, the viral DNA must enter
the cell in order for the virus to replicate
(not the viral protein).
Heredity, Genes, and DNA
The structure of DNA was determined in
1953 by Watson and Crick, using
information from X-ray crystallography
and ideas on hydrogen bonding in the α
helix of proteins.
Heredity, Genes, and DNA
DNA is a double helix with the sugarphosphate backbones on the outside of
the molecule.
The bases are on the inside; hydrogen
bonds are formed between purines and
pyrimidines on opposite chains.
The base pairing is very specific: A
always pairs with T and G with C.
Figure 4.5 The structure of DNA (Part 1)
Figure 4.5 The structure of DNA (Part 2)
Heredity, Genes, and DNA
Complementary base pairing suggested
the mechanism for DNA replication.
The DNA molecule separates, and each
strand becomes a template for
synthesis of a new strand:
• Semiconservative replication—
one strand of parental DNA is
conserved in each progeny DNA
molecule.
Figure 4.6 Semiconservative replication of DNA
Heredity, Genes, and DNA
Experimental support for
semiconservative DNA replication:
Meselson and Stahl grew E. coli in
medium labeled with the heavy isotope
15N.
The heavier DNA could be separated
from light DNA (with 14N) by equilibrium
ultracentrifugation in a CsCl solution.
Heredity, Genes, and DNA
E. coli grown with 15N were transferred
to 14N medium and allowed to replicate
once more.
The DNA from these bacteria was
intermediate in weight.
Figure 4.7 Experimental demonstration of semiconservative replication
Heredity, Genes, and DNA
Further evidence: an enzyme purified
from E. coli (DNA polymerase) could
catalyze DNA replication in vitro.
In the presence of DNA to act as a
template, DNA polymerase directed the
incorporation of nucleotides into a
complementary DNA molecule.
Expression of Genetic Information
Genes determine the structure of
proteins.
The information in DNA is specified by
the order of the four bases.
Proteins are made up of 20 different
amino acids, the sequence of which
determines the protein function.
Expression of Genetic Information
The first direct link between a genetic
mutation and alteration in a protein was
made in 1957.
Patients with inherited sickle-cell anemia
had hemoglobin that differed from
normal hemoglobin by a single amino
acid substitution.
Expression of Genetic Information
The next step was determining the
relationship between DNA and
proteins.
The simplest explanation: the order of
nucleotides in DNA specifies the order
of amino acids in proteins (colinearity).
Mutations (changes in the nucleotide
sequence) should lead to
corresponding changes in the protein.
Expression of Genetic Information
Using E. coli, Yanofsky and colleagues
mapped a series of mutations in the
gene that encodes an enzyme for
tryptophan synthesis.
The relative positions of the amino acid
alterations were the same as those of
the corresponding mutations.
Expression of Genetic Information
Although DNA appeared to specify the
order of amino acids in proteins, it did
not necessarily follow that DNA itself
directs protein synthesis.
DNA is located in the nucleus of
eukaryotic cells, but protein synthesis
occurs in the cytoplasm.
Expression of Genetic Information
RNA was the likely intermediate,
because of its similarity to DNA.
RNA is single-stranded, its sugar is
ribose, and it contains uracil (U) instead
of thymine (T), but this does not affect
base-pairing.
Figure 4.9 Synthesis of RNA from DNA
Expression of Genetic Information
The pathway for the flow of genetic
information:
DNA → RNA → Protein
is now known as the central dogma of
molecular biology.
RNA is synthesized from DNA templates
(transcription); proteins are synthesized
from RNA templates (translation).
Expression of Genetic Information
RNA polymerase catalyzes synthesis of
messenger RNA (mRNA) from a DNA
template.
Ribosomal RNA (rRNA) is a component
of ribosomes, sites of protein synthesis.
Transfer RNAs (tRNAs) serve as
adaptor molecules that align amino
acids along the mRNA template.
Expression of Genetic Information
Each amino acid is attached by a specific
enzyme to its appropriate tRNA.
Base pairing between the tRNA and a
complementary sequence on the mRNA
directs the attached amino acid to its
correct position on the mRNA template.
Figure 4.10 Function of transfer RNA
Expression of Genetic Information
How can four nucleotide bases specify
the sequence of 20 amino acids?
The nucleotides are used as triplets to
encode the different amino acids: the
genetic code.
Expression of Genetic Information
Evidence for the triplet code came from
bacteriophage T4 with mutations in a
gene called rII.
Mutants with the addition of one or two
bases always exhibited the mutant
phenotype.
Changes in three bases frequently led to
the wild-type phenotype.
Figure 4.11 Genetic evidence for a triplet code
Expression of Genetic Information
Assigning nucleotide triplets to their
corresponding amino acids was done
using in vitro systems that could carry
out protein synthesis (in vitro
translation).
The systems contain ribosomes, amino
acids, tRNAs, enzymes, and synthetic
mRNA with known base sequences.
Figure 4.12 The triplet UUU encodes phenylalanine
Expression of Genetic Information
All 64 possible triplets (called codons)
were assigned in this way.
61 specify an amino acid; three are stop
codons that signal the termination of
protein synthesis.
The code is degenerate: many amino
acids are specified by more than one
codon.
Table 4.1 The Genetic Code
Expression of Genetic Information
With few exceptions, all organisms
utilize the same genetic code—strong
support for the conclusion that all
present-day cells evolved from a
common ancestor.
Expression of Genetic Information
Some viruses contain RNA instead of
DNA.
How viral RNA replicates was
determined using RNA bacteriophages
of E. coli.
These viruses encode an enzyme that
catalyzes synthesis of RNA from an
RNA template (RNA-directed RNA
synthesis).
Expression of Genetic Information
Most animal viruses replicate in this way,
but one group (RNA tumor viruses)
requires DNA synthesis in infected
cells.
These viruses (now called retroviruses)
replicate via a DNA intermediate, called
a DNA provirus.
Figure 4.13 Reverse transcription and retrovirus replication
Expression of Genetic Information
This hypothesis was initially met with
disbelief because it reverses the central
dogma.
Later, an enzyme that catalyzes
synthesis of DNA from an RNA
template (reverse transcription) was
discovered.
Key Experiment, Ch. 4, p. 125 (2)
Expression of Genetic Information
Reverse transcription has other broad
implications.
It occurs in cells and is critical for
replicating the ends of eukaryotic
chromosomes, and is frequently
responsible for transposition of DNA
from one chromosomal location to
another.
Expression of Genetic Information
Reverse transcriptases are used
experimentally to generate DNA copies
of any RNA molecule.
This has allowed study and manipulation
of eukaryotic mRNAs.
Recombinant DNA
Recombinant DNA technology allows
scientists to isolate, sequence, and
manipulate individual genes from any
type of cell.
It has enabled detailed molecular studies
of the structure and function of
eukaryotic genes and genomes, and
revolutionized our understanding of cell
biology.
Recombinant DNA
Restriction endonucleases: enzymes
that cleave DNA at specific sequences.
First identified in bacteria, where they
provide defense against the entry of
foreign DNA.
Bacteria have a variety of restriction
endonucleases that cleave DNA at
more than 100 distinct recognition
sites.
Table 4.2 Recognition Sites of Representative Restriction Endonucleases
Recombinant DNA
EcoRI recognizes the sequence
GAATTC.
This sequence is present at five sites in
DNA of bacteriophage λ, so the DNA is
digested into 6 fragments ranging from
3.6 to 21.2 kb long.
1 kilobase (kb) = 1000 base pairs
Recombinant DNA
The fragments can be separated by gel
electrophoresis:
A gel of agarose or polyacrylamide is
placed between two electrodes and the
sample is added to the gel. Nucleic acids
are negatively charged so they migrate
toward the positive electrode.
Smaller molecules move more rapidly,
allowing the fragments to be separated
by size.
Figure 4.14 EcoRI digestion and gel electrophoresis of l DNA
Recombinant DNA
The order of restriction fragments can
also be determined, and maps of
restriction sites generated.
Detailed restriction maps of viral DNA
molecules have been produced.
DNA fragments can also be isolated for
sequencing.
Figure 4.15 Restriction maps of l and adenovirus DNAs
Recombinant DNA
For larger DNA molecules, restriction
endonuclease digestion alone does not
provide enough resolution.
For example, the human genome would
yield more than 500,000 EcoRI
fragments, which can’t be separated by
gel electrophoresis.
Recombinant DNA
Purified DNA fragments can be obtained
by molecular cloning.
In molecular cloning, a DNA fragment
is inserted into a DNA molecule (a
vector) that can replicate
independently in a host cell.
The result is a recombinant molecule
or molecular clone.
Recombinant DNA
Fragments of human DNA can be cloned
in plasmid vectors: small circular DNA
molecules that can replicate
independently in bacteria.
Recombinant plasmids with human DNA
inserts can be introduced into E. coli,
where they replicate along with the
bacteria to yield millions of copies of
plasmid DNA.
Figure 4.16 Generation of a recombinant DNA molecule
Recombinant DNA
The fragment can be isolated from the
plasmid DNA by restriction
endonuclease digestion and gel
electrophoresis.
This allows a pure fragment of human
DNA to be analyzed and further
manipulated.
Recombinant DNA
Restriction endonucleases cleave
recognition sequences at staggered
sites, leaving single-stranded tails that
can associate with each other by
complementary base pairing.
DNA ligase can then seal the ends
permanently.
Figure 4.17 Joining of DNA molecules
Recombinant DNA
Synthetic DNA “linkers” containing
desired restriction endonuclease sites
can be added to the ends of any DNA
fragment.
This allows virtually any fragment of
DNA to be ligated to a vector and
isolated as a molecular clone.
Recombinant DNA
RNA can also be cloned.
RNA is copied using reverse
transcriptase. The resulting DNA
(cDNA) is ligated to a vector DNA.
This allows mRNA to be isolated as a
molecular clone, and the noncoding
sequences in eukaryotic genes
(introns) can be explored.
Figure 4.18 cDNA cloning
Recombinant DNA
Plasmids are often used for cloning DNA
inserts up to a few thousand base pairs
long.
Plasmids have an origin of replication
(ori)—the DNA sequence that signals
the host DNA polymerase to start
replication.
Figure 4.19 Cloning in plasmid vectors (Part 1)
Figure 4.19 Cloning in plasmid vectors (Part 2)
Figure 4.19 Cloning in plasmid vectors (Part 3)
Recombinant DNA
Plasmid vectors also carry genes that
confer resistance to antibiotics, so
bacteria carrying the plasmids can be
selected for.
Recombinant DNA
Bacteriophage λ vectors can accommodate
larger DNA fragments.
Sequences not needed for virus replication
are removed and replaced with unique
restriction sites for insertion of cloned
DNA.
Recombinant DNA
The recombinant molecules are then put
into E. coli, where they replicate to yield
millions of progeny phages containing a
single DNA insert.
Recombinant DNA
For even larger fragments of DNA, five
major types of vectors are used:
1. Cosmid vectors contain
bacteriophage λ sequences, origins of
replication, and genes for antibiotic
resistance, so they are able to replicate
as plasmids in bacterial cells.
Recombinant DNA
2. Bacteriophage P1 vectors allow
recombinant molecules to be packaged
in vitro into P1 phage particles and
replicated as plasmids in E. coli.
3. P1 artificial chromosome (PAC)
vectors also have bacteriophage P1
sequences, but are introduced directly
as plasmids into E. coli.
Recombinant DNA
4. Bacterial artificial chromosome
(BAC) vectors are derived from a
naturally occurring plasmid of E. coli
(the F factor).
5. Yeast artificial chromosome (YAC)
vectors have yeast origins of replication
and other sequences that allow them to
replicate as linear chromosome-like
molecules in yeast cells.
Table 4.3 Vectors for Cloning Large Fragments of DNA
Recombinant DNA
Nucleotide sequencing aids the study of
protein structure, gene sequences that
regulate expression, and gene function.
DNA sequencing is usually done with
automated systems.
Recombinant DNA
The basic method is based on
premature termination of DNA
synthesis.
DNA synthesis is initiated with a
synthetic primer.
Dideoxynucleotides are included along
with the normal nucleotides. They are
labeled with different fluorescent dyes.
Recombinant DNA
The dideoxynucleotides stop DNA
synthesis because no 3 OH group is
available for addition of the next
nucleotide.
The fragments are separated by gel
electrophoresis, and a laser beam
excites the fluorescent dyes.
The data is analyzed by computer.
Figure 4.20 DNA sequencing
Recombinant DNA
Next-generation sequencing allows
DNA to be sequenced faster and less
expensively.
These methods simultaneously
determine sequences of millions of
templates by parallel sequencing
reactions in flow cells.
Recombinant DNA
Molecular cloning is also used to make
large amounts of protein for study.
Many proteins in cells are present in
small amounts and can’t be purified.
Cloned genes can be used to engineer
vectors that lead to high levels of gene
expression in bacteria or eukaryotic
cells.
Recombinant DNA
In bacteria, cDNA is cloned into a
plasmid or phage vector (an
expression vector).
Inserted genes can be expressed at
levels as high as 10% of the total
bacterial protein.
Figure 4.21 Expression of cloned genes in bacteria (Part 1)
Figure 4.21 Expression of cloned genes in bacteria (Part 2)
Recombinant DNA
Expression in eukaryotic cells instead of
bacteria may be needed, (e.g., if
posttranslational modification of the
protein is required).
Cloned genes are inserted into virus
vectors.
Detection of Nucleic Acids and Proteins
Detection of specific nucleic acids and
proteins is important for a variety of
studies:
•
Analysis of gene expression
•
Localization of proteins to subcellular
organelles
Detection of Nucleic Acids and Proteins
To isolate large amounts of a single
DNA molecule, an alternative to
molecular cloning is the polymerase
chain reaction (PCR), developed in
1988.
DNA polymerase is used in vitro for
repeated replication of a defined
segment of DNA.
Detection of Nucleic Acids and Proteins
A specific region of DNA can be
amplified if the nucleotide sequence
surrounding the region is known.
Two primers are designed to initiate
DNA synthesis in opposite directions at
the desired point.
The synthetic primers are 15–20 bases
long.
Detection of Nucleic Acids and Proteins
The reaction starts by heating the
template DNA to 95°C to separate the
strands.
The DNA polymerase used (Taq
polymerase) is a heat-stable enzyme
from Thermus aquaticus, a bacterium
that lives in hot springs.
Detection of Nucleic Acids and Proteins
The temperature is then lowered to allow
primers to pair and DNA synthesis
proceeds.
Two DNA molecules are synthesized
from one template.
The cycle can be repeated multiple
times, with a two-fold increase in DNA
for each cycle.
Figure 4.22 Amplification of DNA by PCR
Detection of Nucleic Acids and Proteins
PCR amplification can be performed
rapidly and automatically.
RNA can also be amplified by PCR if
reverse transcriptase is used to
synthesize a cDNA copy first.
Detection of Nucleic Acids and Proteins
If enough of a gene sequence is known
so that primers can be made, PCR can
detect small amounts of specific DNA
or RNA in complex mixtures.
The only DNA molecules that will be
amplified are those containing
sequences complementary to the
primers used.
Detection of Nucleic Acids and Proteins
Real-time PCR can determine amounts
of specific DNA in a sample.
A fluorescent dye that binds to doublestranded DNA, and only fluoresces
when bound, is incorporated.
Fluorescence is monitored after each
cycle of PCR, and the intensity of
fluorescence is indicative of the amount
of DNA that has been amplified.
Detection of Nucleic Acids and Proteins
The amount of DNA amplified after a
given number of cycles is determined
by the amount of template that was
initially present.
The sensitivity of real-time PCR has
made it important for a variety of
applications, including analysis of gene
expression in cells and tissues.
Detection of Nucleic Acids and Proteins
The key to detection of specific nucleic
acid sequences is base pairing.
In nucleic acid hybridization, DNA
strands are separated by high
temperatures; when cooled, they reform double-stranded molecules by
complementary base pairing.
Detection of Nucleic Acids and Proteins
Cloned DNA can be labeled with
radioactive nucleotides or nucleotides
modified to fluoresce.
This labeled DNA is then used as a
probe that hybridizes with
complementary DNA or RNA in
complex mixtures.
Figure 4.23 Detection of DNA by nucleic acid hybridization
Detection of Nucleic Acids and Proteins
Southern blotting:
DNA is digested with a restriction
endonuclease, and the fragments
separated by gel electrophoresis.
The gel is then overlaid with a
nitrocellulose or nylon membrane to
which the DNA fragments are transferred
(blotted). The filter is then incubated with
a labeled probe.
Figure 4.24 Southern blotting
Detection of Nucleic Acids and Proteins
Northern blotting is used for detection
of RNA instead of DNA.
It is often used in studies of gene
expression, for example, to determine
whether specific mRNAs are present.
Detection of Nucleic Acids and Proteins
Recombinant DNA libraries are
collections of clones that contain all the
genomic or mRNA sequences of a
particular cell type.
A genomic library of human DNA can be
made by cloning random DNA
fragments of about 15 kb in a
bacteriophage λ vector.
Figure 4.25 Screening a recombinant library by hybridization
Detection of Nucleic Acids and Proteins
Any gene for which a probe is available
can be isolated from a recombinant
library.
cDNA clones can be used as probes to
isolate corresponding genomic clones.
Or a gene cloned from one species (e.g.,
mouse) can be used to isolate a related
gene from a different species (e.g.,
human).
Detection of Nucleic Acids and Proteins
In situ hybridization can be used to
detect homologous DNA or RNA
sequences in cell extracts,
chromosomes, or intact cells.
Hybridization of fluorescent probes to
specific cells or subcellular structures is
analyzed by microscopic examination.
Figure 4.26 Fluorescence in situ hybridization
Detection of Nucleic Acids and Proteins
Antibodies can be used as protein
probes.
Antibodies are proteins produced by
immune system cells (B lymphocytes)
that react against foreign molecules
(antigens).
Different antibodies recognize unique
antigens.
Detection of Nucleic Acids and Proteins
Antibodies can be generated by
inoculation of an animal with any
foreign protein.
Monoclonal antibodies can be
produced by culturing clonal lines of B
lymphocytes from immunized animals
(usually mice).
Detection of Nucleic Acids and Proteins
Two common methods using antibodies:
1. Immunoblotting (Western blotting)
Proteins are separated by size by SDSpolyacrylamide gel electrophoresis
(SDS-PAGE).
Detection of Nucleic Acids and Proteins
The negatively charged detergent
sodium dodecyl sulfate (SDS)
denatures the protein and gives it an
overall negative charge.
The proteins will migrate towards the
positive electrode and are then reacted
with labeled antibodies.
Figure 4.27 Immunoblotting (Part 1)
Figure 4.27 Immunoblotting (Part 2)
Figure 4.27 Immunoblotting (Part 3)
Detection of Nucleic Acids and Proteins
Antibodies can be used to visualize
proteins in intact cells.
Cells can be stained with antibodies
labeled with fluorescent dyes or tags
visible by electron microscopy.
Figure 4.28 Immunofluorescence
Gene Function in Eukaryotes
In classical genetics, gene function has
been revealed by the altered
phenotypes of mutant organisms.
It is now possible to study the function of
a cloned gene directly by reintroducing
it into eukaryotic cells.
Gene Function in Eukaryotes
Gene function can be studied by
introducing cloned DNA into plant and
animal cells (gene transfer).
Methods were initially developed using
infectious viral DNA; thus it is called
transfection.
Figure 4.29 Introduction of DNA into animal cells
Gene Function in Eukaryotes
Other methods include:
•
Direct microinjection into the nucleus
•
Coprecipitation of DNA with calcium
phosphate to form small particles that
are taken up by the cells
•
Incorporation of DNA into liposomes
that fuse with the plasma membrane
•
Expose cells to brief electric pulses to
open pores in the plasma membrane
(electroporation)
Gene Function in Eukaryotes
In most of the cells, the DNA is
transported to the nucleus, and is
transcribed for several days: transient
expression.
In about 1% of cells, the foreign DNA is
integrated into the genome and
transferred to progeny cells at cell
division.
Gene Function in Eukaryotes
If the transfected DNA contains a
selectable marker, the stably
transformed cells can be isolated and
studied.
Animal viruses, especially retroviruses,
can be used as vectors to introduce
cloned DNAs into a wide variety of cell
types.
Figure 4.30 Retroviral vectors (Part 1)
Figure 4.30 Retroviral vectors (Part 2)
Gene Function in Eukaryotes
Cloned genes can also be introduced
into the germ line of multicellular
organisms.
Mice that carry foreign genes
(transgenic mice) are produced by
microinjection of cloned DNA into the
pronucleus of a fertilized egg.
Figure 4.31 Production of transgenic mice
Gene Function in Eukaryotes
Embryonic stem (ES) cells are also
used to get cloned genes into mice.
Cloned DNA is introduced into ES cells
in culture, then transformed cells are
introduced back into mouse embryos.
The offspring are chimeric: a mixture of
cells that arise from normal and
transfected embryonic cells.
Figure 4.32 Introduction of genes into mice via embryonic stem cells (Part 1)
Figure 4.32 Introduction of genes into mice via embryonic stem cells (Part 2)
Gene Function in Eukaryotes
Cloned DNAs can also be introduced
into plant cells.
Since many plants can be regenerated
from a single cell, transgenic plants are
easily established.
Many economically important plants,
including corn, tomatoes, soybeans
and potatoes, are transgenic varieties.
Gene Function in Eukaryotes
Classically, gene function has been
studied by observing altered
phenotypes of mutant organisms.
Now, specific mutations can be
introduced into cloned DNA (in vitro
mutagenesis) to study gene function in
eukaryotes.
Gene Function in Eukaryotes
In vitro mutagenesis allows detailed
characterization of the functional roles
of both regulatory and protein-coding
sequences of cloned genes.
The basic method uses synthetic
oligonucleotides to generate changes
in a DNA sequence.
Figure 4.33 Mutagenesis with synthetic oligonucleotides
Gene Function in Eukaryotes
To determine the role of a cloned gene,
activity of the normal gene copy must
be eliminated.
Homologous recombination: the
mutated copy of the cloned gene
replaces the normal gene copy.
Mutations that inactivate (knockout) the
cloned gene are introduced in place of
the normal gene copy.
Figure 4.34 Gene inactivation by homologous recombination
Gene Function in Eukaryotes
Genes can be inactivated in mouse
embryonic stem cells, which can grow
into transgenic mice.
The mice yield progeny with mutated
copies of the gene on both homologous
chromosomes.
Effects of gene inactivation can then be
studied in the context of the intact
animal.
Figure 4.35 Production of mutant mice by homologous recombination in ES cells
Gene Function in Eukaryotes
Cells can be cultured from mouse
embryos containing the mutated gene
copies, so gene function can also be
studied in cell culture.
The function of more than 7000 mouse
genes have been investigated in this
way.
Gene Function in Eukaryotes
Methods have also been developed to
conditionally knockout genes in specific
mouse tissues, allowing the function of
a gene to be studied in a defined cell
type.
Gene Function in Eukaryotes
The CRISPR/Cas system is a new
approach for introducing targeted gene
mutations into mammalian cells.
Specific target sequences are
recognized by a synthetic RNA
molecule, and Cas (CRISPRassociated system) proteins cleave the
targeted DNA.
Gene Function in Eukaryotes
Plasmids expressing a guide RNA
(gRNA) with sequences homologous to
the target gene and the Cas9 nuclease
are introduced into cells, along with a
mutated copy of the gene.
Figure 4.36 Gene targeting by CRISPR/Cas
Gene Function in Eukaryotes
Other approaches interfere with gene
expression or function.
Antisense nucleic acids are RNA or
single-stranded DNA complementary to
the mRNA of the gene of interest
(antisense).
They hybridize with the mRNA and block
its translation into protein.
Figure 4.37 Inhibition of gene expression by antisense RNA or DNA
Gene Function in Eukaryotes
RNA interference (RNAi) was first
discovered in C. elegans. Fire and Mello
found that injection of double-stranded
RNA inhibited expression of a gene with a
complementary mRNA sequence.
Double-stranded RNA resulted in extensive
degradation of the target mRNA, whereas
single-stranded antisense RNA had only
a minimal effect.
Gene Function in Eukaryotes
When double-stranded RNAs are
introduced into cells, they are cleaved
into short interfering RNAs (siRNAs) by
an enzyme called Dicer.
The siRNAs associate with a complex of
proteins known as the RNA-induced
silencing complex (RISC), where
mRNA is cleaved.
Figure 4.38 RNA interference
Key Experiment, Ch. 4, p. 151 (3)
Gene Function in Eukaryotes
The discovery of RNA interference
demonstrated a role for doublestranded RNAs in gene regulation.
This has been developed into a powerful
experimental tool for inhibiting
expression of target genes.
Gene Function in Eukaryotes
It is sometimes possible to interfere
directly with protein function:
• Microinject antibodies that block the
activity of the protein.
Gene Function in Eukaryotes
•
Introduce cloned DNA that encodes
mutant proteins (dominant
inhibitory or dominant negative
mutants) that interfere with the
function of their normal counterparts.
Figure 4.39 Direct inhibition of protein function