Welcome Each of
You to My
Molecular Biology
Class
Molecular Biology of the Gene,
5/E --- Watson et al,(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the Genome
Part III,Expression of the Genome
Part IV,Regulation
Part V,Methods
3/22/05
Part V,Methods
Ch 20,Techniques
of Molecular Biology
Ch 21,Model
Organisms
CHAPTER 21
Model Organisms
?Molecular Biology Course
Model Organisms
?Fundamental problems are
solved in the simplest and
most accessible system in
which the problem can be
addressed.
?These organisms are called
model organisms.
Some Important Model
Organisms
?Escherichia coli and its phage (the
T phage and phage λ)
?Baker’s yeast Saccharomyces
cerevisiae
?The nematode Caenorhabditis
elegans
?The fruit fly Drosophila
melanogaster
?The house mouse Mus musculus
Features of Model Systems
?The availability of powerful
tools of traditional and
molecular genetics.
?The study of each model
system attracted a critical
mass of investigators,
(Ideas,methods,tools and
strains could be shared)
HOW to choose a model
organism?
It depends on what question is
being asked,When studying
fundamental issues of molecular
biology,simpler unicellular
organisms or viruses are
convenient,For developmental
questions,more complicated
organisms should be used.
Model 1,
BACTERIOPHAGE
CHAPTER 15 The Genetic Code
5/08/05
Bacteriophage (Viruses)
?The simplest system
?Their genomes are
replicated only after being
injected into a host cell.
?The genomes can
recombine during these
infections.
Figure Bacteriophage
Each phage attaches to a
specific cell surface
molecule (usually a
protein) and so only cells
bearing that,receptor”
can be infected by a given
phage.
Two Basic Types
1.Lytic phage,eg,T phage
infect a bacterial cell
DNA replication
coat proteins expression
host cell lysed to release the
new phage
Figure 21-1
The lytic growth cycle
2,Temperate phage,
eg,Phage λ
?Lysogeny (溶源途径 )—the phage
genome integrated into the
bacterial genome and
replicated passively as part of
the host chromosome,coat
protein genes not expressed,
? The phage is called a prophage.
? Daughter cells are lysogens.
Figure 21-2
The lysogenic
cycle of a
bacteriophage
?The lysogenic state can switch
to lytic growth,called
induction.
Excision of the prophage DNA
DNA replication
Coat proteins expression
Lytic growth
Figure 16-24
Growth and
induction of λ
lysogen
Assays of Phage Growth
?Progagate phage:
by growth on a suitable
bacterial host in liquid
culture.
?Quantify phage:
plaque (嗜菌斑 ) assay
Ba
cte
rio
ph
ag
e
Progagate phage
?Find a suitable
host cell that
supports the
growth of the virus.
?The mixture of
viruses and
bacteria are filtered
through a bacterial-
proof filter.
Quantify phage
?Phage are mixed with and adsorb to
bacterial cells.
?Dilute the mix.
?Add dilutions to,soft agar” (contain
many uninfected bacterial cells).
?Poured onto a hard agar base.
?Incubated to allow bacterial growth
and phage infection.
Soft
agarHard agar
a petri dish
This circle-of-death produces a
hole or PLAQUE in a lawn of
living cells,These plaques can
be easily seen and counted so
that the numbers of virus can
be quantitated,
As the viruses
replicate and
are released,
they spread
and infect the
nearby cells.
The Single-Step Growth Curve
Ba
cte
rio
ph
ag
e
Figure 21-4
Latent period-
the time lapse
between
infection and
release of
progeny.
Burst size-the
number of
phage released
The Single-Step Growth Curve
?It reveals the life cycle of a
typical lytic phage.
?It reveals the length of time it
takes a phage to undergo one
round of lytic growth,and also
the number of progeny phage
produced per infected cell.
Method
1,Phage were mixed with bacterial
cells for 10 minutes,(Long enough for
adsorption but too short for further
infection progress.)
2,The mixture is diluted by 10,000,
(Only those cells that bound phage in
the initial incubation will contribute to
the infected population; progeny phage
produced from those infections will not
find host cells to infect.)
3,Incubate the dilution,At
intervals,a sample can be
removed from the mixture and
the number of free phage counted
using a plaque assay.
Phage Crosses and
Complementation Tests
Ba
cte
rio
ph
ag
e
Mixed infection,a single
cell is infected with two
phage particles at once.
Mixed infection (co-infection)
1,It allows one to perform phage
crosses.
If two different mutants of the
same phage co-infect a cell,
recombination can occur between
the genomes,The frequency of
this genetic exchange can be used
to order genes on the genome.
2,It allows one to assign
mutations to complementation
groups,
If two different mutant phage co-infect
the same cell and as a result each
provides the function that the other
was lacking,the two mutations must be
in different genes (complementation
groups),If not,the two mutations are
likely located in the same gene.
Transduction and
Recombinant DNA
Ba
cte
rio
ph
ag
e
?During infection,a phage might pick
up a piece of bacterial DNA (mostly
happens when a prophage excises form
the bacterial chromosome),
?The resulting recombinant phage can
transfer the bacterial DNA from one
host to another,known as specialized
transduction.
eg,Phage λ
Model 2,
BACTERIA
CHAPTER 15 The Genetic Code
5/08/05
Features of bacteria
?a single chromosome
?a short generation time
?convenient to study
genetically
Assays of Bacteria Growth
Ba
cte
ria
?Bacteria can be grow in liquid or
on solid (agar) medium.
?Bacterial cells are large enough
to scatter light,allowing the
growth of a bacterial culture to be
monitored in liquid culture by the
increase in optical density (OD).
?Bacterial
cells can grow
exponentially
when not
over-crowded,
called
exponential
phase.
?As the population increase to high
numbers of cells,the growth rate
slows,called stationary phase.
Figure 21-5 Bacteria growth curve
Quantify bacteria
? Dilute the culture.
? Plate the cells on solid medium in a
petri dish.
? Single cells grow into colonies; count
the colonies.
? Knowing how many colonies are on the
plate and how much the culture was
diluted makes it possible to calculate
the concentration of cells in the
original culture.
Bacteria Exchange DNA by:
Ba
cte
ria
?Sexual Conjugation
?Phage-Mediated
Transduction
?DNA-Mediated
Transformation
We use genetic change to:
?Map mutations.
?Construct strains with multiple
mutations.
?Build partially diploid strains
for distinguishing recessive
from dominant mutations and
for carrying out cis-trans
analyses.
?Sexual Conjugation
Plasmids,autonomously
replicating DNA elements in
bacteria.
Some plasmids are capable of
transferring themselves from
one cell to another,
eg,F-factor (fertility plasmid of
E.coli)
?F+ cell,cell
harboring an F-
factor.
?Hfr strain,a
strain harboring
an integrated F-
factor in its
chromosome.
?F’-lac, an F-
factor
containing the
lactose operon.Figure 21-6
?F’ plasmid is a fertility
plasmid that contains a
small segment of
chromosomal DNA.
?F’-factors can be used to
create partially diploid
strains.
?eg,F’-lac
?F-factor-mediated
conjugation is a replicative
process,The products of
conjugating are two F+ cells.
?The F-factor can undergo
conjugation only with other
E.coli strains.
? Some plasmids can transfer DNA
to a wide variety of unrelated
strains,called promiscuous
conjugative plasmids.
? They provide a convenient
means for introducing DNA into
bacteria strains that can’t
undergo genetic exchange.
?Phage-mediated
transduction
?Generalized transduction,A
fragment of chromosomal DNA
is packaged instead of phage
DNA,When such a phage
infects a cell,it introduces the
segment of chromosomal DNA
to the new cell.
?Specialized transduction
Figure 21-7
Phage-
mediated
generalized
transduction
?DNA-mediated
transformation
?Some bacterial species can
take up and incorporate linear,
naked DNA into their own
chromosome by
recombination.
?The cells must be in a
specialized state known as
“genetic competence”.
Bacterial Plasmids Can Be
Used as Cloning Vectors
Ba
cte
ria
?Plasmid,circular DNA in
bacteria that can replicate
autonomously,
?Plasmids can serve as
vectors for bacterial DNA as
well as foreign DNA.
?DNA should be inserted
without impairing the plasmid
replication.
Transposons Can Be Used
to Generate Insertional
Mutations and Gene and
Operon Fusions
Ba
cte
ria eg1,Transposons that integrate
into the chromosome with low-
sequence specificity can be used
to generate a library of
insertional mutations on a
genome-wide basis.
Figure 21-8 Transposon-generated
insertional mutagenesis
Insertional mutations generated by
transposons have two advantages over
traditional mutations.
?The insertion of a transposon
into a gene is more likely to
result in complete
inactivation of the gene.
?Having inactivated the gene,
the inserted DNA is easy to
isolate and clone that gene.
eg2,Gene and operon fusions
created by transopsons
Promoter-less lacZ
Reporter gene
Gene fusion,a fusion in which the reporter
is joined both transcriptionally and
translationally to the target gene.
Figure
21-9
Studies on the Molecular
Biology of Bacteria Have
Been Enhanced by
Recombinant DNA
Technology,Whole-
Genome Sequencing,and
Transcriptional Profiling
Ba
cte
ria
Biochemical Analysis Is
Especially Powerful in
Simple Cells with Well-
Developed Tools of
Traditional and Molecular
Genetics
Ba
cte
ria
?Large quantities of bacterial cells
can be grown in a defined and
homogenous physiological state.
?It is easier to purify protein
complexes harboring precisely
engineered alterations or to
overproduce and obtain individual
proteins in large quantities.
?It is much simpler to carry out
DNA replication,gene
transcription,protein synthesis,
etc,in bacteria than in higher
cells.
Bacteria Are Accessible to
Cytological Analysis
Ba
cte
ria
Despite their simplicity and the
absence of membrane-bound cellular
compartments,bacteria are accessible
to the tools of cytology,such as:
?Immunofluorescence microscopy
?Fluorescence microscopy
?Fluorescence in situ hybridization (FISH)
Phage and Bacteria Told
Us Most of the
Fundamental Things about
the Gene
Ba
cte
ria
There are countless examples
where fundamental processes
of life were understood by
choosing these simplest of
systems.
Model 3,
BAKER’S YEAST
Saccharomyces cerevisiae
CHAPTER 15 The Genetic Code
5/08/05
Features of S,cerevisiae
?Have small genomes
?Can be grown rapidly in the lab
?Central characteristics,
? they contain a discrete (不连续的 )
nucleus with multiple linear
chromosomes packaged into chromatin;
? their cytoplasm includes a full
spectrum of intracellular organelles
and cytoskeletal structures.
The Existence of Haploid and
Diploid Cells Facilitate Genetic
Analysis of S,cerevisiae
Sa
cc
ha
ro
m
yc
es
ce
re
vi
si
ae
S,Cerevisiae can grow in either a
haploid state (one copy of each
chromosome) or diploid state (two
copies of each chromosome).
Conversion between the two states is
mediated by mating (haploid to
diploid) and sporulation (diploid to
haploid).
Figure 21-10
S,Cerevisiae
exist in
three forms,
two haploid
cell types,a
and а,and
the diploid
product of
mating
between
these two.
Application in the Lab
?Genetic complementation,by
mating two haploid strains,each
contains one of the two mutations
whose complementation is being
tested,
?Testing the function of an individual
gene,mutations can be made in
haploid cells in which there is only a
single copy of that gene.
Generating Precise Mutations
in Yeast Is EasySac
ch
ar
om
yc
es
ce
re
vi
si
ae
When linear DNA with ends
homologous to any given region of
the genome is introduced into S,
cerevisiae cells,very high rates of
homologous recombination are
observed resulting in the
transformation.
Figure 21-11
Recombinational
transformation in yeast
This property can
be used to make
precise changes
within the
genome,allowing
very detailed
questions to be
elucidated.
S,Cerevisiae Has a Small,
Well-Characterized Genome
? S,Cerevisiae was the first eukaryotic
organism to have its genome entirely
sequenced,(1996)
? 1.3X106 bp,approximately 6,000 genes.
? The availability of the complete
genome sequence has allowed
“genome-wide” approaches to studies
of this organism.
Sa
cc
ha
ro
m
yc
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ce
re
vi
si
ae
S,Cerevisiae Cells Change
Shape as They GrowSac
ch
ar
om
yc
es
ce
re
vi
si
ae
?S,Cerevisiae divides by budding,
The bud grows until it reaches a
size approximately equal to the size
of the mother cell and is released
from it.
?The emergence of a new bud is
tightly connected to the initiation of
DNA replication.
Start replicating its genome
Chromosome
segregation
Figure 21-12
The mitotic
cell cycle in
yeast
Model 4, THE
NEMATODE WORM,
Caenorhabditis
elegans
CHAPTER 15 The Genetic Code
4/22/05
Caenorhabditis elegans
?Suitable characteristics:
?Rapid generation time
?Hermaphrodite(雌雄同体的 )
reproduction producing
large numbers of,self-
progeny”
?Sexual reproduction so
that genetic stocks could
be constructed
?A small number of
transparent cells so that
development could be
followed directly
C.elegans Has a Very
Raplid Life Cycle
?C.elegans is cultured on
petri dishes,fed a simple
diet of bacteria and grow
well at 25° C,
Ca
en
or
ha
bd
iti
s
el
eg
an
s
Figure 21-13
The lifecycle of
the C,elegans
12 h
Eggs
Juvenile
Adult
Death
12h
40h
15d
?Dauer
?Forming under stressful
condition
?Resistant to environmental
stresses
?Living many months while
waiting for environmental
conditions to improve
C,elegans Is Composed of
Relatively Few,Well Studied
Cell LineagesC
ae
no
rh
ab
di
tis
el
eg
an
s
Figure 21-14 a
Figure 21-14 b The body plan of the
wrom
gonad:生殖腺 oocyte:卵母细胞
uterus:子宫 vulva:阴孔 pharynx:咽
intestine:肠 anus:肛门
?Mutations that disrupt the
formation of the vulva form
a,bag of worms” (the
hatched worms devour
their mother and become
trapped inside her skin).
?The genes are components
of a highly conserved
receptor tyrosine kinase
signaling pathway that
controls cell proliferation.
?Mutations that inactivate this
pathway eliminate vulva
development.
?Mutations that activate this
pathway cause overproliferation
of the vulva precursor cells.
The cell Death Pathway Was
Discovered in C,elegans
?Cell death is under genetic
control (a mutated ced
gene).
?Analysis of the ced mutants
showed that the cell
commits suicide,In males,
a cell known as the linker
cell is killed by its neighbor.
Ca
en
or
ha
bd
iti
s
el
eg
an
s
RNAi Was Discovered in
C,elegans
?RNAi silencing
Ca
en
or
ha
bd
iti
s
el
eg
an
s
Enzyme Dicer makes siRNAs
siRNAs direct a complex called RISC
to repress gene in three ways
Translational
inhibition
Motifying
promotersDigesting mRNA
Figure 17-30
?In 1998,RNAi was discovered
in C,elegans,which is
significant in two respects:
?RNAi appears to be universal.
?Experimental investigation
reveals the molecular
mechanisms.
Model 5,THE
FRUIT FLY,
Drosophila
melanogaster
CHAPTER 15 The Genetic Code
4/22/05
? In 1908,Thomas Hunt Morgan
and his research associates at
Columbia University placed
rotting fruit on the window
ledge of their laboratory,Among
the menagerie of creatures that
were captured,the fruit fly
emerged as the animal of choice.
Drosophila Has a Rapid
Life Cycle
Dr
oso
ph
ila
m
el
an
og
ast
er
Figure 21-15
The rapid life
cycle of
Drosophila
?The growth of the imaginal
disks,arising from invaginations
of epidermis in mid-stage
embryos.
Figure 21-16
Imaginal
disks in
Drosophila
? There is disks for appendages,
eyes,antennae,the mouthparts,
and genitalia.
? Disks are composed of fewer
than 100 cells in the embryo but
thousands of cells in mature
larvae.
? The wing imaginal disk has
become an important model
system for the control of
complex patterning processes
by gradients of secreted
signaling molecules.
The First Genome Maps Were
Produced in Drosophila
?Useful features of the flies in
experimental research,
? Fecundity
? Rapid life cycle
? Four chromosomes (two large
autosomes,a smaller X,and a
very small fourth chromosome)
? Polytene chromosomes
Dr
oso
ph
ila
m
el
an
og
ast
er
Endoreplication in the absence
of mitosis generates enlarged
chromosomes in some tissues of
the fly
Figure 21-17 Polytene chromosomes
?Two major discoverise by the
Morgan lab in 1910,
? Mendel’s first law,Genes are
located on chromosomes,and
each gene is composed of two
alleles that assort independently
during meiosis,
? Mendel’s second law,Genes
located on separate
chromosomes segregate
independently
?By the 1930s,extensive
genetic maps were produced
that identified the relative
positions of numerous genes,
(the distances between
linked genes mapped by
recombination frequencies)
?Large-scale,genetic screens”
are performed by feeding
adult males a mutagen which
cause mutations,and then
mating them with normal
females.(A variety of method
used to study these mutations)
? Method one
? Bridges used polytene chormosomes
to determine a physical map of the
Drosophila genome.
? Bridges identified 5000,bands” on
the four chromosomes and
established a correlation between
the bands and the locations of
genetic loci,
For example
Female fruit flies that contain the
white mutation and a small
deletion in the other X
chromosome,which removes
polytene bands 3C2-3C3,exhibit
white eyes,This type of analysis
led to the conclusion that the
white gene is located between
polytene bands 3C2-3C3 on the
X chromosome
?Method two
?Balancer chromosomes
contain inversions
Figure 21-16
?Such balancers fail to
undergo recombination with
the native chromosome,
Thus,it is possible to
maintain fruit flies that
contain recessive,lethal
mutations,
Genetic Mosaics Permit the
Analysis of Lethal Genes in Adult
Files
?Mosaics are animals that
contain small patches of
mutant tissue in a generally
“normal” genetic background.
?The most spectacular genetic
mosaics are gyandromorphs.
Dr
oso
ph
ila
m
el
an
og
ast
er
Figure 21-19
Gyandromorphs
?Rarely,one of the two X
chromosomes is lost at the
first mitotic division.
?Sexual identity in flies is
determined by the number of
X chromosomes,(two-
female; one-male)
?Suppose that one of the X
chromosomes contains the
recessive white allele,Then
one half of the fly,the male
half,has white eyes,While
the other female half,has
red eyes,
The yeast FLP Recombinase
Permits the Efficient Production
of Genetic Mosaics
?The frequency of mitotic
recombination was greatly
enhanced by the use of the
FLP recombinase from yeast.
?FLP recognizes FRT and
catalyzes DNA rearrangement.
Dr
oso
ph
ila
m
el
an
og
ast
er
?FRT sequences were inserted
near the centromere of each of
the four chromosomes using P-
element transformation.
?Heterozygous flies contain a null
allele in gene Z on one chromoso-
me and a wild-type copy of that
gene on the other.
?In transgenic strains that
contain FLP protein coding
sequence under the control
of heat-inducible hsp70
promoter,FLP is synthesized
upon heat shock.
?FLP binds to the FRT motifs in the
two homologs containing gene Z and
catalyze mitotic recombination.
Figure 21-20 FLP-FRT
It Is Easy to Create Transgenic
Fruit Flies that Carry Foreign DNA
?P-elements are transposable
DNA that can cause hybrid
dysgenesis (杂交不育 ).(how? )
Dr
oso
ph
ila
m
el
an
og
ast
er
Figure 21-21 hybrid dysgenesis
? The F1
progeny are
often sterile,
when mating
females from
the,M” strain
with males
from the,P”
strain.
?P-elements encode both a
repressor of transposition and
a transposase that promotes
mobilization.
?The repressor is expressed in
the developing P eggs,Thus M
eggs lack the repressor that
inhibits p-element mobilization.
?Sometimes the P-elements
insert into genes that are
essential for the development
of progenitors of the gametes
(配偶子 ),and,as a result,the
adult flies derived from the
these matings are sterile.
?P-elements can be used as
vectors in the transformation
of the fly embryos.
Figure 21-22
?A full length P-element
transposon is 3 kb in length,
It contains inverted repeats
at the termini that are
essential for excision and
insertion.
?Recombinant DNA is inserted
into defective P-elements
that lack the internal genes
encoding repressor and
transposase.
?Transposase is injected along
with the recombination P-
element vector.
? The recombinant P-elements
insert into random positions in
the pole cells,
? The amount of recombination p-
element and transposase is
calibrated so that,on average,a
given pole cell receives just a
single integrated p-element.
?The embryos are allowed to
develop into adults and then
mated with tester strains.
?The recombinant P-element
contains a,marker” gene
such as white+.
?P-element transformation
is routinely used to identify
regulatory sequences.
?It can also be used to
examine protein coding
genes in various genetic
backgrounds.
Model 6,THE
HOUSE MOUSE,
Mus musculus
CHAPTER 15 The Genetic Code
4/22/05
?The mouse is an excellent
model for human development
and disease,although,the life
cycle of the mouse is slow by
the standard of the nematode
worm and fruit fly.
The predominance
of the mouse model
? The mouse provides the link
between the basic principles,
discovered in simpler creatures
like worms and flies,and human
disease.
? The chromosome complement is
similar between the mouse and
human (autosomomes and X,Y
sex chromosomes)
? Extended regions of a given
mouse chromosome contain
“homologous” regions of the
corresponding human
chromosomes,(more than 85%
of the mouse genes are
correspond to human genes.)
Mouse Embryonic Development
Depends on Stem Cells
?The first obvious diversification
of cell types is at the 16-cell
stage,called the morula (桑椹胚 ).
? The cells in outer regions of the
morula develop into the placenta
(胎盘 ).
? Cells in internal regions generate
the inner cell mass (ICM) which
is the prime source of embryonic
stem cells.
M
us
m
us
cu
lu
s
?At the 64-cell stage the mouse
embryo,called a blastocyst (胚泡 ),
is ready for implantation,
Interactions between the
blastocyst and uterine wall lead
to the formation of the plancenta.
?Then the embryo enters gastrulat-
ion (原肠胚 ),and the ICM forms all
three germ layers,endoderm (内胚
层 ),mesoderm (中胚层 ),ectoderm
(外胚层 ).
? The first stage in gastrulation is
the subdivision of the ICM into two
cell lays,an inner hypoblast (内胚层 )
and an outer epiblast (外胚层 ).
? Then a groove called primitive
streak (原条 ) forms along the
length of the epiblast and the
cells that migrate into the
groove form the internal
mesoderm,The anterior end of
the primitive streak is the node.
?Shortly thereafter,a fetus
emerges that contains a
brain,a spinal cord,and
internal organs (eg,heart
and liver).
Figure 21-23
overview of
mouse
embryogenesis
It is Easy to Introduce Foreign
DNA into the Mouse Embryo
?Create transgenic mice by
microinjection method,
First,Inject DNA into the egg
pronucleus.
Second,place the embryos into
the oviduct (输卵管 ) of a female
mouse.
Third,the injected DNA
integrates at random positions
in the genome.
M
us
m
us
cu
lu
s
Figure 21-24
?Germline transformation,
the offspring of transgenic
mice also contain the foreign
recombinant DNA.
Figure 21-25
?A transgenic
strain of mice was
created that
contains a portion
of the Hoxb-2
regulatory region
attached to a lacZ
report gene,There
are two bands of
staining detected
in the hindbrain
region of 10.5 day
embryos.
Homologous Recombination
Permits the Selective Ablation
of Individual Genes
?The single most powerful
method of mouse transgenesis
is the ability to disrupt,or
“knock out,” single genetic loci,
This permits the creation of
mouse models for human
disease.
M
us
m
us
cu
lu
s
?Gene disruption experiments
? They are done with embryonic
stem (ES) cells.
? A recombinant DNA is created
that contains a mutant form of
the gene of interest.
? The method is used to create a
cell line lacking any given gene.
?The process of the experiment
First,designing the recombination
vector,It contains the modified
target gene,the NEO gene
(downstream of the target gene),
the region of homology with the
host cell chromosome
(downstream of and flanking NEO)
and a marker (TK,gene for
thymidine kinase).
Second,transform the vector into
ES cells.
Third,select for NEO,Only the
cells which undergo double
recombination with the host cell
chromosome can survive in the
neomycin containing medium,
Fourth,select against TK,If
illicit recombination occurs,TK
gene will frequently be
contained,In this case,the
cells which undergo illicit
recombination will die in the
GANC containing medium.
Fifth,harvest the homologous
recombination ES cells and
inject them into the ICM of
normal blastocysts.
Sixth,insert the hybrid embryo
into the oviduct of a host mouse
and allowed to develop to term.
Figure 21-26
Mice Exhibit Epigenetic
Inheritance
?Parental imprinting,only one
of the two alleles for certain
genes is active,because the
other copy of is selectively
inactivated either in the
developing sperm cell or the
developing egg.
M
us
m
us
cu
lu
s
Figure 21-27
imprinting in
the mouse
See the
detail in
chapter 17
You to My
Molecular Biology
Class
Molecular Biology of the Gene,
5/E --- Watson et al,(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the Genome
Part III,Expression of the Genome
Part IV,Regulation
Part V,Methods
3/22/05
Part V,Methods
Ch 20,Techniques
of Molecular Biology
Ch 21,Model
Organisms
CHAPTER 21
Model Organisms
?Molecular Biology Course
Model Organisms
?Fundamental problems are
solved in the simplest and
most accessible system in
which the problem can be
addressed.
?These organisms are called
model organisms.
Some Important Model
Organisms
?Escherichia coli and its phage (the
T phage and phage λ)
?Baker’s yeast Saccharomyces
cerevisiae
?The nematode Caenorhabditis
elegans
?The fruit fly Drosophila
melanogaster
?The house mouse Mus musculus
Features of Model Systems
?The availability of powerful
tools of traditional and
molecular genetics.
?The study of each model
system attracted a critical
mass of investigators,
(Ideas,methods,tools and
strains could be shared)
HOW to choose a model
organism?
It depends on what question is
being asked,When studying
fundamental issues of molecular
biology,simpler unicellular
organisms or viruses are
convenient,For developmental
questions,more complicated
organisms should be used.
Model 1,
BACTERIOPHAGE
CHAPTER 15 The Genetic Code
5/08/05
Bacteriophage (Viruses)
?The simplest system
?Their genomes are
replicated only after being
injected into a host cell.
?The genomes can
recombine during these
infections.
Figure Bacteriophage
Each phage attaches to a
specific cell surface
molecule (usually a
protein) and so only cells
bearing that,receptor”
can be infected by a given
phage.
Two Basic Types
1.Lytic phage,eg,T phage
infect a bacterial cell
DNA replication
coat proteins expression
host cell lysed to release the
new phage
Figure 21-1
The lytic growth cycle
2,Temperate phage,
eg,Phage λ
?Lysogeny (溶源途径 )—the phage
genome integrated into the
bacterial genome and
replicated passively as part of
the host chromosome,coat
protein genes not expressed,
? The phage is called a prophage.
? Daughter cells are lysogens.
Figure 21-2
The lysogenic
cycle of a
bacteriophage
?The lysogenic state can switch
to lytic growth,called
induction.
Excision of the prophage DNA
DNA replication
Coat proteins expression
Lytic growth
Figure 16-24
Growth and
induction of λ
lysogen
Assays of Phage Growth
?Progagate phage:
by growth on a suitable
bacterial host in liquid
culture.
?Quantify phage:
plaque (嗜菌斑 ) assay
Ba
cte
rio
ph
ag
e
Progagate phage
?Find a suitable
host cell that
supports the
growth of the virus.
?The mixture of
viruses and
bacteria are filtered
through a bacterial-
proof filter.
Quantify phage
?Phage are mixed with and adsorb to
bacterial cells.
?Dilute the mix.
?Add dilutions to,soft agar” (contain
many uninfected bacterial cells).
?Poured onto a hard agar base.
?Incubated to allow bacterial growth
and phage infection.
Soft
agarHard agar
a petri dish
This circle-of-death produces a
hole or PLAQUE in a lawn of
living cells,These plaques can
be easily seen and counted so
that the numbers of virus can
be quantitated,
As the viruses
replicate and
are released,
they spread
and infect the
nearby cells.
The Single-Step Growth Curve
Ba
cte
rio
ph
ag
e
Figure 21-4
Latent period-
the time lapse
between
infection and
release of
progeny.
Burst size-the
number of
phage released
The Single-Step Growth Curve
?It reveals the life cycle of a
typical lytic phage.
?It reveals the length of time it
takes a phage to undergo one
round of lytic growth,and also
the number of progeny phage
produced per infected cell.
Method
1,Phage were mixed with bacterial
cells for 10 minutes,(Long enough for
adsorption but too short for further
infection progress.)
2,The mixture is diluted by 10,000,
(Only those cells that bound phage in
the initial incubation will contribute to
the infected population; progeny phage
produced from those infections will not
find host cells to infect.)
3,Incubate the dilution,At
intervals,a sample can be
removed from the mixture and
the number of free phage counted
using a plaque assay.
Phage Crosses and
Complementation Tests
Ba
cte
rio
ph
ag
e
Mixed infection,a single
cell is infected with two
phage particles at once.
Mixed infection (co-infection)
1,It allows one to perform phage
crosses.
If two different mutants of the
same phage co-infect a cell,
recombination can occur between
the genomes,The frequency of
this genetic exchange can be used
to order genes on the genome.
2,It allows one to assign
mutations to complementation
groups,
If two different mutant phage co-infect
the same cell and as a result each
provides the function that the other
was lacking,the two mutations must be
in different genes (complementation
groups),If not,the two mutations are
likely located in the same gene.
Transduction and
Recombinant DNA
Ba
cte
rio
ph
ag
e
?During infection,a phage might pick
up a piece of bacterial DNA (mostly
happens when a prophage excises form
the bacterial chromosome),
?The resulting recombinant phage can
transfer the bacterial DNA from one
host to another,known as specialized
transduction.
eg,Phage λ
Model 2,
BACTERIA
CHAPTER 15 The Genetic Code
5/08/05
Features of bacteria
?a single chromosome
?a short generation time
?convenient to study
genetically
Assays of Bacteria Growth
Ba
cte
ria
?Bacteria can be grow in liquid or
on solid (agar) medium.
?Bacterial cells are large enough
to scatter light,allowing the
growth of a bacterial culture to be
monitored in liquid culture by the
increase in optical density (OD).
?Bacterial
cells can grow
exponentially
when not
over-crowded,
called
exponential
phase.
?As the population increase to high
numbers of cells,the growth rate
slows,called stationary phase.
Figure 21-5 Bacteria growth curve
Quantify bacteria
? Dilute the culture.
? Plate the cells on solid medium in a
petri dish.
? Single cells grow into colonies; count
the colonies.
? Knowing how many colonies are on the
plate and how much the culture was
diluted makes it possible to calculate
the concentration of cells in the
original culture.
Bacteria Exchange DNA by:
Ba
cte
ria
?Sexual Conjugation
?Phage-Mediated
Transduction
?DNA-Mediated
Transformation
We use genetic change to:
?Map mutations.
?Construct strains with multiple
mutations.
?Build partially diploid strains
for distinguishing recessive
from dominant mutations and
for carrying out cis-trans
analyses.
?Sexual Conjugation
Plasmids,autonomously
replicating DNA elements in
bacteria.
Some plasmids are capable of
transferring themselves from
one cell to another,
eg,F-factor (fertility plasmid of
E.coli)
?F+ cell,cell
harboring an F-
factor.
?Hfr strain,a
strain harboring
an integrated F-
factor in its
chromosome.
?F’-lac, an F-
factor
containing the
lactose operon.Figure 21-6
?F’ plasmid is a fertility
plasmid that contains a
small segment of
chromosomal DNA.
?F’-factors can be used to
create partially diploid
strains.
?eg,F’-lac
?F-factor-mediated
conjugation is a replicative
process,The products of
conjugating are two F+ cells.
?The F-factor can undergo
conjugation only with other
E.coli strains.
? Some plasmids can transfer DNA
to a wide variety of unrelated
strains,called promiscuous
conjugative plasmids.
? They provide a convenient
means for introducing DNA into
bacteria strains that can’t
undergo genetic exchange.
?Phage-mediated
transduction
?Generalized transduction,A
fragment of chromosomal DNA
is packaged instead of phage
DNA,When such a phage
infects a cell,it introduces the
segment of chromosomal DNA
to the new cell.
?Specialized transduction
Figure 21-7
Phage-
mediated
generalized
transduction
?DNA-mediated
transformation
?Some bacterial species can
take up and incorporate linear,
naked DNA into their own
chromosome by
recombination.
?The cells must be in a
specialized state known as
“genetic competence”.
Bacterial Plasmids Can Be
Used as Cloning Vectors
Ba
cte
ria
?Plasmid,circular DNA in
bacteria that can replicate
autonomously,
?Plasmids can serve as
vectors for bacterial DNA as
well as foreign DNA.
?DNA should be inserted
without impairing the plasmid
replication.
Transposons Can Be Used
to Generate Insertional
Mutations and Gene and
Operon Fusions
Ba
cte
ria eg1,Transposons that integrate
into the chromosome with low-
sequence specificity can be used
to generate a library of
insertional mutations on a
genome-wide basis.
Figure 21-8 Transposon-generated
insertional mutagenesis
Insertional mutations generated by
transposons have two advantages over
traditional mutations.
?The insertion of a transposon
into a gene is more likely to
result in complete
inactivation of the gene.
?Having inactivated the gene,
the inserted DNA is easy to
isolate and clone that gene.
eg2,Gene and operon fusions
created by transopsons
Promoter-less lacZ
Reporter gene
Gene fusion,a fusion in which the reporter
is joined both transcriptionally and
translationally to the target gene.
Figure
21-9
Studies on the Molecular
Biology of Bacteria Have
Been Enhanced by
Recombinant DNA
Technology,Whole-
Genome Sequencing,and
Transcriptional Profiling
Ba
cte
ria
Biochemical Analysis Is
Especially Powerful in
Simple Cells with Well-
Developed Tools of
Traditional and Molecular
Genetics
Ba
cte
ria
?Large quantities of bacterial cells
can be grown in a defined and
homogenous physiological state.
?It is easier to purify protein
complexes harboring precisely
engineered alterations or to
overproduce and obtain individual
proteins in large quantities.
?It is much simpler to carry out
DNA replication,gene
transcription,protein synthesis,
etc,in bacteria than in higher
cells.
Bacteria Are Accessible to
Cytological Analysis
Ba
cte
ria
Despite their simplicity and the
absence of membrane-bound cellular
compartments,bacteria are accessible
to the tools of cytology,such as:
?Immunofluorescence microscopy
?Fluorescence microscopy
?Fluorescence in situ hybridization (FISH)
Phage and Bacteria Told
Us Most of the
Fundamental Things about
the Gene
Ba
cte
ria
There are countless examples
where fundamental processes
of life were understood by
choosing these simplest of
systems.
Model 3,
BAKER’S YEAST
Saccharomyces cerevisiae
CHAPTER 15 The Genetic Code
5/08/05
Features of S,cerevisiae
?Have small genomes
?Can be grown rapidly in the lab
?Central characteristics,
? they contain a discrete (不连续的 )
nucleus with multiple linear
chromosomes packaged into chromatin;
? their cytoplasm includes a full
spectrum of intracellular organelles
and cytoskeletal structures.
The Existence of Haploid and
Diploid Cells Facilitate Genetic
Analysis of S,cerevisiae
Sa
cc
ha
ro
m
yc
es
ce
re
vi
si
ae
S,Cerevisiae can grow in either a
haploid state (one copy of each
chromosome) or diploid state (two
copies of each chromosome).
Conversion between the two states is
mediated by mating (haploid to
diploid) and sporulation (diploid to
haploid).
Figure 21-10
S,Cerevisiae
exist in
three forms,
two haploid
cell types,a
and а,and
the diploid
product of
mating
between
these two.
Application in the Lab
?Genetic complementation,by
mating two haploid strains,each
contains one of the two mutations
whose complementation is being
tested,
?Testing the function of an individual
gene,mutations can be made in
haploid cells in which there is only a
single copy of that gene.
Generating Precise Mutations
in Yeast Is EasySac
ch
ar
om
yc
es
ce
re
vi
si
ae
When linear DNA with ends
homologous to any given region of
the genome is introduced into S,
cerevisiae cells,very high rates of
homologous recombination are
observed resulting in the
transformation.
Figure 21-11
Recombinational
transformation in yeast
This property can
be used to make
precise changes
within the
genome,allowing
very detailed
questions to be
elucidated.
S,Cerevisiae Has a Small,
Well-Characterized Genome
? S,Cerevisiae was the first eukaryotic
organism to have its genome entirely
sequenced,(1996)
? 1.3X106 bp,approximately 6,000 genes.
? The availability of the complete
genome sequence has allowed
“genome-wide” approaches to studies
of this organism.
Sa
cc
ha
ro
m
yc
es
ce
re
vi
si
ae
S,Cerevisiae Cells Change
Shape as They GrowSac
ch
ar
om
yc
es
ce
re
vi
si
ae
?S,Cerevisiae divides by budding,
The bud grows until it reaches a
size approximately equal to the size
of the mother cell and is released
from it.
?The emergence of a new bud is
tightly connected to the initiation of
DNA replication.
Start replicating its genome
Chromosome
segregation
Figure 21-12
The mitotic
cell cycle in
yeast
Model 4, THE
NEMATODE WORM,
Caenorhabditis
elegans
CHAPTER 15 The Genetic Code
4/22/05
Caenorhabditis elegans
?Suitable characteristics:
?Rapid generation time
?Hermaphrodite(雌雄同体的 )
reproduction producing
large numbers of,self-
progeny”
?Sexual reproduction so
that genetic stocks could
be constructed
?A small number of
transparent cells so that
development could be
followed directly
C.elegans Has a Very
Raplid Life Cycle
?C.elegans is cultured on
petri dishes,fed a simple
diet of bacteria and grow
well at 25° C,
Ca
en
or
ha
bd
iti
s
el
eg
an
s
Figure 21-13
The lifecycle of
the C,elegans
12 h
Eggs
Juvenile
Adult
Death
12h
40h
15d
?Dauer
?Forming under stressful
condition
?Resistant to environmental
stresses
?Living many months while
waiting for environmental
conditions to improve
C,elegans Is Composed of
Relatively Few,Well Studied
Cell LineagesC
ae
no
rh
ab
di
tis
el
eg
an
s
Figure 21-14 a
Figure 21-14 b The body plan of the
wrom
gonad:生殖腺 oocyte:卵母细胞
uterus:子宫 vulva:阴孔 pharynx:咽
intestine:肠 anus:肛门
?Mutations that disrupt the
formation of the vulva form
a,bag of worms” (the
hatched worms devour
their mother and become
trapped inside her skin).
?The genes are components
of a highly conserved
receptor tyrosine kinase
signaling pathway that
controls cell proliferation.
?Mutations that inactivate this
pathway eliminate vulva
development.
?Mutations that activate this
pathway cause overproliferation
of the vulva precursor cells.
The cell Death Pathway Was
Discovered in C,elegans
?Cell death is under genetic
control (a mutated ced
gene).
?Analysis of the ced mutants
showed that the cell
commits suicide,In males,
a cell known as the linker
cell is killed by its neighbor.
Ca
en
or
ha
bd
iti
s
el
eg
an
s
RNAi Was Discovered in
C,elegans
?RNAi silencing
Ca
en
or
ha
bd
iti
s
el
eg
an
s
Enzyme Dicer makes siRNAs
siRNAs direct a complex called RISC
to repress gene in three ways
Translational
inhibition
Motifying
promotersDigesting mRNA
Figure 17-30
?In 1998,RNAi was discovered
in C,elegans,which is
significant in two respects:
?RNAi appears to be universal.
?Experimental investigation
reveals the molecular
mechanisms.
Model 5,THE
FRUIT FLY,
Drosophila
melanogaster
CHAPTER 15 The Genetic Code
4/22/05
? In 1908,Thomas Hunt Morgan
and his research associates at
Columbia University placed
rotting fruit on the window
ledge of their laboratory,Among
the menagerie of creatures that
were captured,the fruit fly
emerged as the animal of choice.
Drosophila Has a Rapid
Life Cycle
Dr
oso
ph
ila
m
el
an
og
ast
er
Figure 21-15
The rapid life
cycle of
Drosophila
?The growth of the imaginal
disks,arising from invaginations
of epidermis in mid-stage
embryos.
Figure 21-16
Imaginal
disks in
Drosophila
? There is disks for appendages,
eyes,antennae,the mouthparts,
and genitalia.
? Disks are composed of fewer
than 100 cells in the embryo but
thousands of cells in mature
larvae.
? The wing imaginal disk has
become an important model
system for the control of
complex patterning processes
by gradients of secreted
signaling molecules.
The First Genome Maps Were
Produced in Drosophila
?Useful features of the flies in
experimental research,
? Fecundity
? Rapid life cycle
? Four chromosomes (two large
autosomes,a smaller X,and a
very small fourth chromosome)
? Polytene chromosomes
Dr
oso
ph
ila
m
el
an
og
ast
er
Endoreplication in the absence
of mitosis generates enlarged
chromosomes in some tissues of
the fly
Figure 21-17 Polytene chromosomes
?Two major discoverise by the
Morgan lab in 1910,
? Mendel’s first law,Genes are
located on chromosomes,and
each gene is composed of two
alleles that assort independently
during meiosis,
? Mendel’s second law,Genes
located on separate
chromosomes segregate
independently
?By the 1930s,extensive
genetic maps were produced
that identified the relative
positions of numerous genes,
(the distances between
linked genes mapped by
recombination frequencies)
?Large-scale,genetic screens”
are performed by feeding
adult males a mutagen which
cause mutations,and then
mating them with normal
females.(A variety of method
used to study these mutations)
? Method one
? Bridges used polytene chormosomes
to determine a physical map of the
Drosophila genome.
? Bridges identified 5000,bands” on
the four chromosomes and
established a correlation between
the bands and the locations of
genetic loci,
For example
Female fruit flies that contain the
white mutation and a small
deletion in the other X
chromosome,which removes
polytene bands 3C2-3C3,exhibit
white eyes,This type of analysis
led to the conclusion that the
white gene is located between
polytene bands 3C2-3C3 on the
X chromosome
?Method two
?Balancer chromosomes
contain inversions
Figure 21-16
?Such balancers fail to
undergo recombination with
the native chromosome,
Thus,it is possible to
maintain fruit flies that
contain recessive,lethal
mutations,
Genetic Mosaics Permit the
Analysis of Lethal Genes in Adult
Files
?Mosaics are animals that
contain small patches of
mutant tissue in a generally
“normal” genetic background.
?The most spectacular genetic
mosaics are gyandromorphs.
Dr
oso
ph
ila
m
el
an
og
ast
er
Figure 21-19
Gyandromorphs
?Rarely,one of the two X
chromosomes is lost at the
first mitotic division.
?Sexual identity in flies is
determined by the number of
X chromosomes,(two-
female; one-male)
?Suppose that one of the X
chromosomes contains the
recessive white allele,Then
one half of the fly,the male
half,has white eyes,While
the other female half,has
red eyes,
The yeast FLP Recombinase
Permits the Efficient Production
of Genetic Mosaics
?The frequency of mitotic
recombination was greatly
enhanced by the use of the
FLP recombinase from yeast.
?FLP recognizes FRT and
catalyzes DNA rearrangement.
Dr
oso
ph
ila
m
el
an
og
ast
er
?FRT sequences were inserted
near the centromere of each of
the four chromosomes using P-
element transformation.
?Heterozygous flies contain a null
allele in gene Z on one chromoso-
me and a wild-type copy of that
gene on the other.
?In transgenic strains that
contain FLP protein coding
sequence under the control
of heat-inducible hsp70
promoter,FLP is synthesized
upon heat shock.
?FLP binds to the FRT motifs in the
two homologs containing gene Z and
catalyze mitotic recombination.
Figure 21-20 FLP-FRT
It Is Easy to Create Transgenic
Fruit Flies that Carry Foreign DNA
?P-elements are transposable
DNA that can cause hybrid
dysgenesis (杂交不育 ).(how? )
Dr
oso
ph
ila
m
el
an
og
ast
er
Figure 21-21 hybrid dysgenesis
? The F1
progeny are
often sterile,
when mating
females from
the,M” strain
with males
from the,P”
strain.
?P-elements encode both a
repressor of transposition and
a transposase that promotes
mobilization.
?The repressor is expressed in
the developing P eggs,Thus M
eggs lack the repressor that
inhibits p-element mobilization.
?Sometimes the P-elements
insert into genes that are
essential for the development
of progenitors of the gametes
(配偶子 ),and,as a result,the
adult flies derived from the
these matings are sterile.
?P-elements can be used as
vectors in the transformation
of the fly embryos.
Figure 21-22
?A full length P-element
transposon is 3 kb in length,
It contains inverted repeats
at the termini that are
essential for excision and
insertion.
?Recombinant DNA is inserted
into defective P-elements
that lack the internal genes
encoding repressor and
transposase.
?Transposase is injected along
with the recombination P-
element vector.
? The recombinant P-elements
insert into random positions in
the pole cells,
? The amount of recombination p-
element and transposase is
calibrated so that,on average,a
given pole cell receives just a
single integrated p-element.
?The embryos are allowed to
develop into adults and then
mated with tester strains.
?The recombinant P-element
contains a,marker” gene
such as white+.
?P-element transformation
is routinely used to identify
regulatory sequences.
?It can also be used to
examine protein coding
genes in various genetic
backgrounds.
Model 6,THE
HOUSE MOUSE,
Mus musculus
CHAPTER 15 The Genetic Code
4/22/05
?The mouse is an excellent
model for human development
and disease,although,the life
cycle of the mouse is slow by
the standard of the nematode
worm and fruit fly.
The predominance
of the mouse model
? The mouse provides the link
between the basic principles,
discovered in simpler creatures
like worms and flies,and human
disease.
? The chromosome complement is
similar between the mouse and
human (autosomomes and X,Y
sex chromosomes)
? Extended regions of a given
mouse chromosome contain
“homologous” regions of the
corresponding human
chromosomes,(more than 85%
of the mouse genes are
correspond to human genes.)
Mouse Embryonic Development
Depends on Stem Cells
?The first obvious diversification
of cell types is at the 16-cell
stage,called the morula (桑椹胚 ).
? The cells in outer regions of the
morula develop into the placenta
(胎盘 ).
? Cells in internal regions generate
the inner cell mass (ICM) which
is the prime source of embryonic
stem cells.
M
us
m
us
cu
lu
s
?At the 64-cell stage the mouse
embryo,called a blastocyst (胚泡 ),
is ready for implantation,
Interactions between the
blastocyst and uterine wall lead
to the formation of the plancenta.
?Then the embryo enters gastrulat-
ion (原肠胚 ),and the ICM forms all
three germ layers,endoderm (内胚
层 ),mesoderm (中胚层 ),ectoderm
(外胚层 ).
? The first stage in gastrulation is
the subdivision of the ICM into two
cell lays,an inner hypoblast (内胚层 )
and an outer epiblast (外胚层 ).
? Then a groove called primitive
streak (原条 ) forms along the
length of the epiblast and the
cells that migrate into the
groove form the internal
mesoderm,The anterior end of
the primitive streak is the node.
?Shortly thereafter,a fetus
emerges that contains a
brain,a spinal cord,and
internal organs (eg,heart
and liver).
Figure 21-23
overview of
mouse
embryogenesis
It is Easy to Introduce Foreign
DNA into the Mouse Embryo
?Create transgenic mice by
microinjection method,
First,Inject DNA into the egg
pronucleus.
Second,place the embryos into
the oviduct (输卵管 ) of a female
mouse.
Third,the injected DNA
integrates at random positions
in the genome.
M
us
m
us
cu
lu
s
Figure 21-24
?Germline transformation,
the offspring of transgenic
mice also contain the foreign
recombinant DNA.
Figure 21-25
?A transgenic
strain of mice was
created that
contains a portion
of the Hoxb-2
regulatory region
attached to a lacZ
report gene,There
are two bands of
staining detected
in the hindbrain
region of 10.5 day
embryos.
Homologous Recombination
Permits the Selective Ablation
of Individual Genes
?The single most powerful
method of mouse transgenesis
is the ability to disrupt,or
“knock out,” single genetic loci,
This permits the creation of
mouse models for human
disease.
M
us
m
us
cu
lu
s
?Gene disruption experiments
? They are done with embryonic
stem (ES) cells.
? A recombinant DNA is created
that contains a mutant form of
the gene of interest.
? The method is used to create a
cell line lacking any given gene.
?The process of the experiment
First,designing the recombination
vector,It contains the modified
target gene,the NEO gene
(downstream of the target gene),
the region of homology with the
host cell chromosome
(downstream of and flanking NEO)
and a marker (TK,gene for
thymidine kinase).
Second,transform the vector into
ES cells.
Third,select for NEO,Only the
cells which undergo double
recombination with the host cell
chromosome can survive in the
neomycin containing medium,
Fourth,select against TK,If
illicit recombination occurs,TK
gene will frequently be
contained,In this case,the
cells which undergo illicit
recombination will die in the
GANC containing medium.
Fifth,harvest the homologous
recombination ES cells and
inject them into the ICM of
normal blastocysts.
Sixth,insert the hybrid embryo
into the oviduct of a host mouse
and allowed to develop to term.
Figure 21-26
Mice Exhibit Epigenetic
Inheritance
?Parental imprinting,only one
of the two alleles for certain
genes is active,because the
other copy of is selectively
inactivated either in the
developing sperm cell or the
developing egg.
M
us
m
us
cu
lu
s
Figure 21-27
imprinting in
the mouse
See the
detail in
chapter 17
