Download Figure 20-6

Chapter 20
Sexual
Reproduction,
Meiosis, and
Genetic
Recombination
Lectures by
Kathleen Fitzpatrick
Simon Fraser University
© 2012 Pearson Education, Inc.
Sexual Reproduction, Meiosis, and
Genetic Recombination
• During asexual reproduction new (genetically
identical) individuals are generated by mitosis
• It can be efficient as long as environmental
conditions don’t change
• However, under changing environmental
conditions, organisms that undergo sexual
reproduction usually have an advantage
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Sexual Reproduction
• Sexual reproduction allows genetic information from
two parents to be mixed together, producing
genetically novel offspring
• Most plants and animals, and many eukaryotic
microorganisms, reproduce sexually
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Sexual Reproduction Produces Genetic
Variety by Bringing Together
Chromosomes from Two Different
Parents
• Sexual reproduction allows for production of an
enormous variety among individuals in a population
• Genetic variety depends on mutations,
unpredictable alterations in DNA base sequence
• They are rare events; beneficial mutations are rarer
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Homologous chromosomes
• Sexually reproducing organisms have cells with two
copies of each type of chromosome, one from each
parent
• The two members of the chromosome pair are
called homologous chromosomes, which look alike
under the microscope
• They carry the same lineup of genes, but these
may vary slightly in base sequence
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Sex chromosomes
• There are two kinds of sex chromosomes,
which determine the gender of the individual
carrying them
• They are generally called X and Y chromosomes,
and differ in appearance, and genetic makeup
(XX  female; XY  male)
• During sexual reproduction the X and Y
chromosomes behave as homologues
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Diploid and haploid
• A cell or organism with two sets of chromosomes
is said to be diploid (2n)
• A cell or organism with one set of chromosomes is
called haploid (n)
• In humans, with 23 pairs of chromosomes, n  23
and 2n  46
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Diploid Cells May Be Homozygous or
Heterozyygous for Each Gene
• A gene locus is the place on a chromosome
that contains the DNA for a particular gene,
which controls a character (trait) in the organism
• Slight variations in the sequence of a gene are
called alleles
• The combination of alleles determines how the
organism will express the character controlled
by the gene
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Figure 20-1
© 2012 Pearson Education, Inc.
Heterozygous and homozygous
• Homozygous individuals have the same two
alleles of a particular gene
• Heterozygous individuals carry two different
alleles of the gene; the appearance of the
organism depends on the relationship between
the alleles
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Dominant and recessive alleles
• In a heterozygous individual, the dominant allele
determines the trait that appears in the individual
• The recessive trait does not show up unless the
individual is homozygous for that allele
• Genotype is the whole genetic makeup of an
individual, whereas the phenotype is the physical
expression of the genotype
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Figure 20-2
© 2012 Pearson Education, Inc.
Gametes Are Haploid Cells Specialized
for Sexual Reproduction
• Gametes are the haploid cells from each parent
that fuse to form a new individual
• In animals and plants, males make sperm and
females make ova (eggs)
• Fertilization, the union of sperm and egg, creates
a zygote
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Variations on gametes
• Parthenogenesis, in which females reproduce
without males, is known but rare
• Unicellular eukaryotes and fungi produce gametes
of identical size rather than sperm and ova; these
gametes are said to differ in mating type
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Meiosis
• Gametes produced by mitosis would be diploid and
would fuse to form a tetraploid (four sets of
chromosomes) offspring
• A different type of cell division is used to produce
gametes with a haploid chromosome content
• This process is meiosis and involves DNA
replication followed by two divisions
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Figure 20-3
© 2012 Pearson Education, Inc.
The Life Cycles of Sexual Organisms
Have Diploid and Haploid Phases
• The life cycles of sexually reproducing organisms is
divided into a diploid (2n) and haploid (1n) phase
• The diploid phase begins at fertilization and
extends to meiosis, whereas the haploid phase
begins at meiosis and ends at fertilization
• Organisms vary greatly in the relative prominence
of haploid and diploid phases
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Fungi are primarily haploid
• Fungi are predominantly haploid, but include a
brief diploid phase that begins with gamete fusion
and ends at meiosis
• Meiosis usually takes place almost immediately
after gamete fusion, so the diploid phase is very
short
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Mosses and ferns have prominent
haploid and diploid phases
• For mosses, the haploid form is larger and more
prominent; in ferns it is the opposite
• In both, gametes develop from preexisting haploid
cells, whereas haploid spores are produced by
meiosis
• This alternation of forms is alternation of
generations
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Alternation of generations
• Haploid spores germinate to give rise to the haploid
form of the plant or alga (gametophyte)
• The haploid form produces gametes by mitosis
• Gametes fuse by fertilization to form the diploid
form, called the sporophyte
• In most plants, the sporophyte generation
predominates
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Alternation of generations in flowering
plants
• In flowering plants, the gametophyte is inside the
flower
• The female gametophyte is called the carpel and
the male gametophyte is the anther
• Meiosis in plants is called sporic meiosis, whereas
in animals it is called gametic meiosis
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Figure 20-4
© 2012 Pearson Education, Inc.
Meiosis Converts One Diploid Cell
into Four Haploid Cells
• Meiosis is preceded by chromosome duplication
and involves two successive divisions
• A diploid nucleus is converted into four haploid
nuclei
• Meiosis I is called the reduction division because it
reduces the chromosome number from diploid to
haploid
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Meiosis I
• Early during meiosis I the chromosomes of each
homologous pair bind together during prophase to
exchange some of their genetic information
• This pairing is called synapsis
• The two chromosomes behave as a unit called a
bivalent (or tetrad) that aligns at the spindle
equator
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Meiosis I and II
• After lining up at the equator, the bivalent splits
so that each member of the pair moves to the
opposite pole of the cell
• Each pole receives only one of each pair, so the
resulting cell is considered haploid
• In meiosis II, the chromatids separate just as in
mitosis
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Meiosis I Produces Two Haploid Cells
That Have Chromosomes Composed
of Sister Chromatids
• The first meiotic division segregates homologues
(and thus the alleles on those homologues) into
different daughter cells
• This separation makes possible the eventual
remixing of different pairs of alleles at fertilization
• This and the exchange of DNA segments is called
genetic recombination
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Prophase I: Homologous
Chromosomes Become Paired and
Exchange DNA
• Prophase I is a particularly long and complex
phase
• It can be divided into five stages: leptotene,
zygotene, pachytene, diplotene, and diakinesis
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Leptotene and zygotene
• The leptotene stage begins with condensation of
chromatin fibers into long, threadlike structures
• At the zygotene stage, condensation continues to
make individual chromosomes distinguishable and
homologues undergo synapsis to form bivalents
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Figure 20-6
© 2012 Pearson Education, Inc.
Pachytene and diplotene
• At the pachytene stage chromosomes condense
dramatically
• DNA segments are exchanged by crossing over
• At the diplotene stage, the homologous
chromosomes begin to separate but remain
attached by connections called chiasmata—the
positions of crossovers
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Figure 20-6
© 2012 Pearson Education, Inc.
Diakinesis
• In diakinesis, chromosomes recondense to their
compacted state
• In some organisms, chromosomes decondense
during diplotene and cells take a break from
meiosis
• In diakinesis chromosomes continue to separate
from their homologues and are only connected by
chiasmata, nucleoli disappear, and the spindle
forms
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Figure 20-6
© 2012 Pearson Education, Inc.
Figure 20-5A
© 2012 Pearson Education, Inc.
Figure 20-6
© 2012 Pearson Education, Inc.
The synaptonemal complex
• Homologous chromosomes are held together by
the synaptonemal complex, an elaborate protein
structure resembling a zipper
• The lateral elements begin to attach to
chromosomes during leptotene
• The central element, which actually joins the
chromosomes together, does not form until
zygotene
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Figure 20-7
© 2012 Pearson Education, Inc.
Figure 20-7A
© 2012 Pearson Education, Inc.
Figure 20-7B
© 2012 Pearson Education, Inc.
Metaphase I: Bivalents Align at the
Spindle Equator
• During metaphase I the bivalents attach via their
kinetochores to spindle microtubules, and migrate
to the spindle equator
• The presence of paired homologues at the equator
is a feature specific to meiosis
• The bivalents are randomly oriented, and
homologues are held together only by chiasmata
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Anaphase I: Homologous
Chromosomes Move to Opposite
Spindle Poles
• As anaphase I begins, homologues separate from
each other and start to migrate toward opposite
spindle poles
• Homologue separation is a fundamental feature of
meiosis
• A protein called shugoshin protects the cohesins at
the centromeres from degradation
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Telophase I and Cytokinesis: Two
Haploid Cells Are Produced
• Telophase I begins when the haploid set of
chromosomes arrives at each spindle pole
• In some cases nuclear envelopes form around the
chromosomes prior to cytokinesis, generating two
haploid cells
• Usually the chromosomes do not decondense
before meiosis II begins
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Figure 20-5 b-d
© 2012 Pearson Education, Inc.
Meiosis II Resembles a Mitotic Division
• A brief interphase may intervene before meiosis II
begins
• Each cell contains one set of replicated
chromosomes, each with two sister chromatids
• The purpose of meiosis II is to divide the sister
chromatids into two newly forming cells
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Meiosis II resembles mitosis
• Prophase II is very brief, and resembles prophase
of mitosis
• In metaphase II chromosomes line up at the
spindle equator as in mitosis, except that there are
only half the normal number of chromosomes
• In anaphase II sister chromatids move to opposite
poles of the cell
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Nondisjunction
• Occasionally an error in segregation called
nondisjunction occurs
• This produces cells that have an extra
chromosome or are missing one, a condition
called aneuploidy
• If aneuploid gametes fuse with normal gametes,
defective embryos are produced that usually die
before birth
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C value
• Through the stages of meiosis, the DNA content of
a cell changes
• The amount of DNA present in a cell is expressed
as C value, where one haploid set of
chromosomes is 1C
• In a diploid cell before replication the ploidy is 2n
and the DNA content is 2C
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C value after replication
• After replication, the DNA is doubled to 4C because
each chromosome consists of two chromatids
• After meiosis I, the chromosome number (ploidy) is
1n and the DNA content is 2C
• After meiosis II, the chromosome number is still 1n,
and the DNA content is reduced to 1C
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Figure 20-8
© 2012 Pearson Education, Inc.
Figure 20-8
© 2012 Pearson Education, Inc.
Sperm and Egg Cells Are Generated
by Meiosis Accompanied by Cell
Differentiation
• In males meiosis converts a diploid spermatocyte
into four haploid spermatids
• After meiosis is complete, the spermatids
differentiate into sperm cells by discarding most of
the cytoplasm, and developing flagella
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Figure 20-9A
© 2012 Pearson Education, Inc.
Video: Meiosis I in Sperm Formation
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Oocyte development
• In females meiosis converts a diploid ooctye into
four haploid cells but only one of the four survives
and gives rise to an egg cell
• The two meiotic divisions divide the cytoplasm
unequally, with one daughter cell receiving the bulk
of the cytoplasm
• The other three very small cells are called polar
bodies
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Figure 20-9B
© 2012 Pearson Education, Inc.
Developing egg cells acquire special
features during meiosis
• Many special features of the egg are acquired
during prophase I, when meiosis I is temporarily
halted to allow for growth
• During this growth phase, the cell develops special
coatings to protect the egg from injury
• Oocytes remain in prophase I until resumption of
meiosis is triggered by a signal
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MPF
• In amphibians, resumption of meiosis is triggered
by progesterone, which causes an increase in MPF
activity
• Progesterone stimulates production of Mos, a
protein kinase that activates a series of kinases,
leading to MPF activation
• In some organisms the second meiotic division
does not occur until fertilization
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Metaphase II arrest
• Metaphase II arrest in vertebrate eggs is triggered
by cytostatic factor (CSF), an inhibitor of the
anaphase-promoting complex
• CSF is inactivated when the egg is fertilized
• The mature egg contains everything needed for
early stages of embryonic development
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Meiosis Generates Genetic Diversity
• Meiosis plays a role in generating genetic diversity
in sexually reproducing populations
• Various combinations of chromosomes are
assembled into gametes to be passed on to the next
generation
• Also, crossing over leads to more combinations of
alleles, generating additional diversity
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Genetic Variability: Segregation and
Assortment of Alleles
• The work of Gregor Mendel laid the foundation for
what is now called Mendelian genetics
• Mendel worked with garden peas and studied
seven readily identifiable characters
• He began by establishing that each of his plant
strains was true-breeding, meaning that plants
produced the same phenotype generation after
generation
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Figure 20-10A
© 2012 Pearson Education, Inc.
Information Specifying Recessive
Traits Can Be Present Without Being
Displayed
• In his first set of experiments Mendel crossfertilized the true-breeding parental plants
(P1 generation) to produce hybrid strains
• The resulting offspring (F1 generation) showed
only one of the parental traits, the dominant trait
• Next, Mendel allowed the F1 hybrids to self-fertilize
and looked at the offspring (F2 generation)
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Figure 20-10B
© 2012 Pearson Education, Inc.
The F2 generation
• For each trait, the F2 generation showed a 3:1 ratio
of dominant to recessive phenotypes
• Mendel allowed the F2 plants to self-fertilize
• The F2 plants with the recessive trait always bred
true, and one-third of the dominant plants bred true
• The remaining F2 plants produced a 3:1 ratio
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Figure 20-11
© 2012 Pearson Education, Inc.
Mendel’s conclusions
• Mendel concluded that information for the
recessive trait must be present in the F1 hybrid
plants even though it was not visible
• An additional experiment, in which F1 hybrids were
backcrossed (crossed to one of the parental
strains), supported this
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Figure 20-12
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Figure 20-12
© 2012 Pearson Education, Inc.
The Law of Segregation States That the
Alleles of Each Gene Separate from
Each Other During Gamete Formation
• Mendel formulated principles, known as Mendel’s
laws of inheritance
• The first was that traits are determined by factors
(genes) present as pairs of determinants (alleles)
• Most scientists believed in a blending theory of
inheritance
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The law of segregation
• Mendel’s law of segregation states that the two
alleles of a gene are distinct entities that separate
from one another during gamete formation
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The Law of Independent Assortment
States That the Alleles of Each Gene
Separate Independently of the Alleles
of Other Genes
• Mendel studied multifactor crosses between plants
that differed in several characters
• He generated F1 hybrid plants and allowed them to
self fertilize
• He found all combinations of traits in the F2
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The Law of Independent Assortment
• Mendel deduced that all combinations of alleles
occurred in the gametes with equal frequency
• The two alleles of each gene segregate
independently of other genes, the law of
independent assortment
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Early Microscopic Evidence
Suggested That Chromosomes
Might Carry Genetic Information
• By 1875 microscopists had identified chromosomes
and fertilization was shown to involve fusion of
sperm and egg nuclei
• The first proposal that chromosomes carry genetic
information was made in 1883
• It was soon realized that the chromosome number
remains constant throughout the development of an
organism
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Rediscovery of Mendel
• Montgomery, Boveri, and Sutton made crucial
observations following the rediscovery of Mendel’s
paper
• Montgomery recognized the existence of
homologous chromosomes
• Boveri observed that each chromosome plays a
unique genetic role with experiments involving
abnormal chromosome numbers in sea urchin eggs
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Rediscovery of Mendel
• Sutton observed that the orientation of each pair
of homologous chromosomes (bivalent) at the
spindle equator during metaphase I is random
• The work of these three scientists helped make
the connection between chromosome behavior
during meiosis and the inheritance of genetic
information
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Chromosome Behavior Explains the
Laws of Segregation and Independent
Assortment
• The chromosome theory of inheritance can be
summarized as follows:
– 1. Nuclei of all cells except the germ line (sperm and
eggs) contain a paternal and a maternal set of
chromosomes
– 2. Chromosomes retain their individuality and are
genetically continuous throughout the life cycle of an
organism
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Chromosome behavior and Mendel’s
laws (continued)
• The chromosome theory of inheritance
– 3. The two sets of homologous chromosomes carry a
similar set of genes
– 4. Maternal and paternal homologues synapse during
meiosis and move to opposite poles of the spindle
– 5. The maternal and paternal members of different
pairs segregate independently during meiosis
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Figure 20-13
© 2012 Pearson Education, Inc.
Figure 20-14
© 2012 Pearson Education, Inc.
The DNA Molecules of
Homologous Chromosomes Have
Similar Base Sequences
• Homologous chromosomes have DNA molecules
whose sequences are nearly identical
• The minor sequence differences create allele
differences and arise from mutations
• DNA homology, the similarity in base sequences,
explains the ability of homologues to undergo
synapsis
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Chromosome pairing
• Chromosomes of some organisms possess DNA
sequences called pairing sites that promote
synapsis between chromosomes
• Proteins in the synaptonemal complex also play a
role in facilitating pairing
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Genetic Variability: Recombination
and Crossing Over
• Segregation and independent assortment of
homologues in meiosis I lead to random
assortment of alleles
• In a diploid organism of genotype Aa Bb, meiosis
will produce gametes in which A assorts with B as
often as with b
• In a case where two genes reside on the same
chromosome, alleles will not assort randomly
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Figure 20-15A
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Figure 20-15B
© 2012 Pearson Education, Inc.
Genes on the same chromosome
• Alleles on the same chromosome tend to
segregate together
• But even in this case, some scrambling of alleles
occurs because of crossing over
• Crossing over involves the exchange of material
during meiosis I when homologues are synapsed
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Figure 20-15C
© 2012 Pearson Education, Inc.
Chromosomes Contain Groups of
Linked Genes That Are Usually
Inherited Together
• Morgan and colleagues first discovered linkage in
the fruit fly
• They began with wild type, the “normal” type of fly
• They had to generate mutations for their genetic
experiments, naturally occurring and X-ray induced
mutations
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Linkage
• An early observation was that not all fruit fly genes
assorted independently; instead some behaved as
though they were physically connected
• In these cases, new combinations of alleles
were rare
• Fruit fly genes can be classified into four linkage
groups
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Linkage groups
• Linkage groups are collections of linked genes
that are usually inherited together
• Morgan realized that the number of linkage groups
was the same as the number of chromosomes in
the fly (n  4)
• He concluded that each linkage group
corresponded to a chromosome
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Homologous Chromosomes Exchange
Segments During Crossing Over
• Morgan found that linkage of linked genes was not
complete
• Though genes assorted together most of the time,
sometimes nonparental combinations would
appear in offspring
• This was called recombination
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Crossing over
• Morgan proposed that homologous chromosomes
can exchange segments through crossing over
• Non-crossover chromosomes are called parental
and those that have crossed over are called
recombinant
• Crossing over takes place during the pachytene
stage of meiotic prophase I; the resulting chiasmata
hold homologues together at metaphase I
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Gene Locations Can Be Mapped by
Measuring Recombination Frequencies
• Morgan and others noticed that recombination
frequency differed for different pairs of genes
• This suggested that crossover frequency was
related to distance between the genes
• They used recombination frequency to determine
the distance between pairs of genes
• This is called genetic mapping
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Figure 20-16
© 2012 Pearson Education, Inc.
Figure 20-16A
© 2012 Pearson Education, Inc.
Figure 20-16B
© 2012 Pearson Education, Inc.
Genetic mapping
• Recombination frequency is used to determine
distances between genes in map units
(centimorgans)
• The percent of nonparental offspring corresponds
to map distance where 1% crossover  1 mu
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Genetic Recombination in Bacteria
and Viruses
• Crossing over is not restricted to sexually
reproducing organisms
• Viruses and bacteria are capable of genetic
recombination
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Co-infection of Bacterial Cells with
Related Bacteriophages Can Lead
to Genetic Recombination
• Genetic recombination between related phages
takes place when bacterial cells are infected by
both types of phage simultaneously
• As the phages replicate in the bacterium, DNA
segments can be exchanged between homologous
regions
• Recombinant phages arise at a frequency
dependent on the distance between the genes
being studied
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Figure 20-17
© 2012 Pearson Education, Inc.
Transformation and Transduction
Involve Recombination with Free DNA
or DNA Brought into Bacterial Cells by
Bacteriophages
• In bacteria, several mechanisms exist for
recombining genetic information
• The ability of bacteria to take up DNA molecules
and incorporate DNA into their genomes is called
transformation
• Transduction involves DNA that has been brought
into a bacterium by a bacteriophage
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Figure 20-18A
© 2012 Pearson Education, Inc.
Transducing phages
• Phages that occasionally incorporate some bacterial
DNA into their progeny phages are called
transducing phages
• Cotransductional mapping involves determining how
frequently two genes are transduced together
• The closer two genes are on a chromosome, the
more likely they will be transduced together
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Figure 20-18B
© 2012 Pearson Education, Inc.
Conjugation Is a Modified Sexual
Activity That Facilitates Genetic
Recombination in Bacteria
• Bacteria also transfer DNA from one cell to another
by conjugation
• It resembles mating in that one bacterium is the
donor (often called “male”) and the other is the
recipient (“female”)
• It usually involves only a portion of the genome and
so does not qualify as true sexual reproduction
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The F Factor
• The F factor enables E. coli cells to act as donors
during conjugation
• It is either an independent replicating plasmid, or a
part of the bacterial chromosome
• Donor cells develop long, hairlike projections called
sex pili, that selectively bind to recipient cells to
form a transient cytoplasmic mating bridge
through which DNA is transferred
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Figure 20-19
© 2012 Pearson Education, Inc.
Figure 20-19A
© 2012 Pearson Education, Inc.
Figure 20-19B
© 2012 Pearson Education, Inc.
DNA transfer
• The donor cell quickly transfers a copy of the
plasmid into the recipient cell (F), transforming
the recipient into a donor cell (F)
• Transfer begins at the origin of transfer
• The donor cell remains F because it retains one
copy of the plasmid
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Figure 20-20A
© 2012 Pearson Education, Inc.
Hfr Cells and Bacterial Chromosome
Transfer
• The F factor can sometimes integrate into the
bacterial chromosome
• This converts the F cell into an Hfr (high
frequency of recombination) cell
• When mated with an F cell, an Hfr cell transfers a
copy of its chromosomal DNA (or part of it) starting
at the origin of transfer
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Figure 20-20b
© 2012 Pearson Education, Inc.
Bacterial chromosome transfer
• Transfer of the entire bacterial chromosome is rare
because it takes about 90 minutes
• Genes located close to the origin of transfer are
most likely to be transferred to the recipient cell
• Once inside the cell, the donor DNA can recombine
with the recipient DNA
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Mapping bacterial chromosomes
• The correlation between the position of a gene on
the chromosome and its likelihood of transfer can
be used to map genes
• A cross is made between Hfr and F strains that
differ in several genetic properties
• After conjugation, cells are plated to allow growth
of recombinant but not parent cells, and frequency
of recombination is calculated
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Figure 20-20C
© 2012 Pearson Education, Inc.
Molecular Mechanism of Homologous
Recombination
• Homologous recombination involves exchange
of genetic information between DNA molecules
with extensive sequence similarity
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DNA Breakage and Exchange Underlies
Homologous Recombination
• Two theories were proposed to explain how
homologous recombination occurs
• The breakage-and-exchange model postulated that
breaks occur in the DNA followed by exchange and
rejoining of the broken segments
• In the copy-choice model genetic recombination
occurs when DNA replication switches from one
homologue to the other
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Experimental evidence for the
breakage-and-exchange model
• In 1961 Meselson and Weigle used phages of the
same type labeled with heavy (15N) or light (14N)
nitrogen
• Co-infecting bacteria with both phages resulted in
some progeny phages containing genes from both
original phages
• The progeny phages contained both isotopes of
nitrogen
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Figure 20-21
© 2012 Pearson Education, Inc.
More evidence in eukaryotes
• Taylor exposed eukaryotic cells to 3H-thymidine
during S phase in the last mitosis prior to meiosis,
then allowed the next S phase to proceed without
3H thymidines generating chromosomes with one
labeled and one unlabeled chromatid
• In the subsequent meiosis chromatids contained a
mixture of radioactive and nonradioactive
segments
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Figure 20-22
© 2012 Pearson Education, Inc.
Homologous Recombination Can
Lead to Gene Conversion
• A simple breakage-exchange model would predict
that genetic recombination should be reciprocal
• This is usually observed, but not always
• Nonreciprocal recombination is called gene
conversion, because one allele appears to be
converted into the other
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Homologous Recombination Is
Initiated by Single-Strand DNA
Exchanges (Holliday Junctions)
• Recombination is not accomplished by cleaving two
double-stranded molecules and then exchanging
and rejoining the cut ends
• Holliday was the first to propose that recombination
is based on the exchange of single DNA strands
between two double-stranded DNA molecules
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Steps of recombination
• One or both strands of the DNA double helix are
cleaved (1)
• A single strand from one molecule invaded a
complementary region of a homologous DNA
double helix, displacing one of the strands (2)
• Strand invasion is catalyzed by the RecA protein
in bacteria and Rad51 in eukaryotes
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Figure 20-23
© 2012 Pearson Education, Inc.
Steps of recombination (continued)
• Localized DNA synthesis and repair generate a
Holliday junction (3, 4)
• Electron microscopy has provided direct evidence
for the existence of Holliday junctions
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Steps of recombination (continued)
• Once the Holliday junction is formed, unwinding
and rewinding the DNA double helix causes
movement of the crossover point (5)
• This is branch migration and can increase the
amount of DNA exchanged
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Cleaving of the Holliday junction
• After branch migration the junction is cleaved and
the broken strands rejoined
• If it is cleaved in one plane, the two DNA molecules
will exhibit crossing over (6a)
• If the junction is cut in the other plane, there is no
crossing over, but there is a noncomplementary
region near the site of the Holliday junction (6b)
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Fate of noncomplementary DNA
• Noncomplementary DNA may be corrected by
repair or left intact
• The net effect of repair can be to convert genes
from one allele to the other
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The Synaptonemal Complex
Facilitates Homologous
Recombination During Meiosis
• The synaptonemal complex appears at the time
when recombination takes place
• Its location between the opposed homologues is
the region where crossover takes place
• Synaptonemal complexes are absent from
organisms that fail to carry out meiotic
recombination
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Homology searching
• Cells ensure that the synaptonemal complex forms
only between homologues
• In homology searching, a single-strand break in one
DNA molecule produces a free strand that invades
another and checks for complementarity
• Only then does the synaptonemal complex develop
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Recombinant DNA Technology and
Gene Cloning
• Recombinant DNA technology has enabled
researchers to isolate and study genes from any
source with greater ease than was thought
possible
• A central feature of the technology is the ability to
produce specific pieces of DNA in large
quantities—this is DNA cloning
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DNA cloning
• DNA cloning is accomplished by the splicing of the DNA of
interest into an element called a cloning vector that can
replicate inside a cell grown in culture
• Usually the vector is a plasmid or virus, grown in bacteria
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The Discovery of Restriction
Enzymes Paved the Way for
Recombinant DNA Technology
• Much of recombinant DNA technology is made
possible by restriction enzymes
• They cleave DNA molecules at specific sequences
called restriction sites
• Those that make staggered cuts in the DNA are
especially useful, because they generate sticky
ends that make it easy to join DNA fragments
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Generating recombinant DNA molecules
• DNA molecules from two sources are treated with
a restriction enzyme that generates sticky ends (1)
• The fragments are mixed together under conditions
that favor base pairing (2)
• The fragments are sealed together by DNA ligase
(3) to produce a recombinant molecule
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Figure 20-24
© 2012 Pearson Education, Inc.
DNA Cloning Techniques Permit
Individual Gene Sequences to Be
Produced in Large Quantities
• Recombinant DNA molecules can be inserted into
a cloning vector that can replicate itself when
introduced into bacteria
• Five steps are typically involved in the process
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1. Insertion of DNA into a Cloning
Vector
• DNA is inserted into a cloning vector, usually a
plasmid or bacteriophage, most of which are
engineered molecules designed for cloning
• Plasmids used as cloning vectors have a variety of
restriction sites and carry antibiotic resistance
genes
• These allow for selection of bacteria containing the
plasmids
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Use of the -galactosidase gene
• Plasmids such as pUC19 have a number of
restriction enzyme sites in a region containing the
lacZ gene, which encodes -galactosidase
• Integration of foreign DNA at one of these sites
disrupts the lacZ gene
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Figure 20-26A
© 2012 Pearson Education, Inc.
How a gene is inserted into a plasmid
vector
• Incubation with the restriction enzyme cuts the
plasmid at a single site in the lacZ gene, making
the DNA linear (1)
• The same restriction enzyme is used to cleave the
DNA to be cloned (2)
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Inserting a gene into a plasmid (continued)
• The cut molecules are incubated under conditions
that favor base pairing (3)
• They are treated with DNA ligase to link the
molecules covalently (4)
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Figure 20-26B
© 2012 Pearson Education, Inc.
Activity: DNA Cloning in a Plasmid Vector
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2. Introduction of the Recombinant
Vector into Bacterial Cells
• If the vector is phage DNA, it is incorporated into
phage particles that are used to infect an
appropriate cell population
• Plasmids are introduced into the medium with
target cells, which take up the plasmid after the
appropriate treatment
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3. Amplification of the Recombinant
Vector in Bacteria
• After cells take up the vector, they are plated on a
medium so that the vector can be replicated or
amplified
• As the bacteria divide, the plasmids also replicate
• Billions can be produced in a short time (less than
half a day)
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Phages
• In the case of phages, the particles with the
recombinant DNA are mixed with bacteria and
placed on a culture medium
• A lawn of bacteria grows on the plate; some cells
are infected by phage, which replicate and cause
lysis of the host cell
• The cycle repeats with nearby cells, producing a
plaque in the lawn
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4. Selection of Cells Containing
Recombinant DNA
• During amplification of the cloning vector,
selection is used to preferentially isolate the cells
that have incorporated the vector
• E.g., bacterial cells that have incorporated pUC19
acquire ampicillin resistance because the plasmid
contains the ampR gene
• This is called a selectable marker
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Selection of cells with recombinant
plasmids
• Not all cells that have acquired plasmids contain
recombinant plasmids (plasmids containing the
DNA insert)
• Cells with recombinant plasmids can be
recognized because the insert disrupts the lacZ
gene, and prevents production of -galactosidase
• Cells with normal pUC19 plasmid stain blue with a
simple color test—with recombinant pUC19, the
cells are white
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Selection of cells with recombinant
phages
• Phage cloning vectors are about 70% as long as
normal phage DNA
• These are too small to be packaged into functional
phage particles
• Only vectors that have the insert DNA added will be
large enough to produce progeny phage particles
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Figure 20-27
© 2012 Pearson Education, Inc.
5. Identification of Clones Containing
the DNA of Interest
• Many cells may be generated that produce many
different types of recombinant DNA
• Recombinant colonies or plaques can be screened to
identify those containing the DNA of interest
• For bacterial clones, DNA is isolated and restriction
enzymes are used to confirm the identity of the
insert DNA
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Genomic and cDNA Libraries Are
Both Useful for DNA Cloning
• In one approach to cloning, the shotgun approach,
an organism’s entire genome is cleaved into a large
number of restriction fragments and inserted into
cloning vectors
• The resulting group of clones, a genomic library,
contains fragments representing most or all of the
genome
• A partial DNA digestion is used to produce
overlapping fragments
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cDNA libraries
• In a second approach, mRNA is copied with
reverse transcriptase, which makes cDNA
(complementary DNA)
• If the entire population of mRNA in a cell is isolated
and copied into cDNA, the result is a cDNA library
• The value of the cDNA library is that it contains only
the sequences that are actively transcribed in the
cell or tissue used to make the library
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Figure 20-28
© 2012 Pearson Education, Inc.
Another advantage to cDNA libraries
• cDNA libraries contain only the gene coding
sequences; there are no introns
• Introns can be large and can make cloned genes too
long for easy DNA manipulation
• Bacteria cannot make the correct protein product
from a gene unless the introns have been removed
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Screening libraries
• Once a library has been constructed, several
approaches to screening for identification of
plasmids or phages that contain genes of interest
can be used
• The type of technique used depends on the prior
knowledge of the gene and the type of library
• If the DNA sequence of the gene is known, a
nucleic acid probe (single stranded DNA or RNA
specific for the desired sequence) can be used
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Screening libraries (continued )
• The nucleic acid probe is labeled with radioactivity
of some other chemical group that allows the probe
to be easily visualized
• Colonies containing sequences complementary to
the probe can be identified by this technique,
called colony hybridization
• DNA is recovered from colonies by isolating the
vector and restriction enzyme digestion
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Figure 20-29
© 2012 Pearson Education, Inc.
Screening approaches based on function
• If the protein product of the gene is known and has
been purified, antibodies that recognize it can be
prepared as probes to check bacteria for the
presence of the protein
• Or, the function of the protein can be measured,
e.g., an enzyme’s activity can be tested
• Techniques like this only work if bacteria are able to
produce the protein; special expression vectors
make this more likely
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Large DNA Segments Can Be Cloned
in YACs and BACs
• Eukaryotic genes are often larger than 30,000 bp,
the upper limit for phage vectors
• In mapping projects, larger clones mean fewer are
needed to cover the whole genome
• Yeast artificial chromosomes (YACs) can
accommodate large inserts; they are “minimal”
chromosomes that contain centromeres, telomeres,
and DNA origins of replication
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YACs
• When the components of the YAC are combined
with foreign DNA, the resulting chromosome will
replicate in yeast and sequence into daughter cells
with each round of cell division
• YACs contain genes that function as selectable
markers and restriction sites for cloning foreign
DNA
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Figure 20-30
© 2012 Pearson Education, Inc.
BACs
• Bacterial artificial chromosomes (BACs) are F
factor derivatives that can hold up to 350,000 bp of
foreign DNA
• They contain a bacterial origin of replication,
antibiotic resistance genes, and insertion sites for
foreign DNA
• One type contains a gene, the product of which
converts sucrose into a toxic substance
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Selection of a BAC with a DNA insert
• Foreign DNA is inserted into the gene (called SacB)
• Bacteria grown in the presence of sucrose will die
unless the BAC they contain has foreign DNA
inserted into SacB, disrupting its function
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PCR Is Widely Used to Clone Genes
from Sequenced Genomes
• In cases where a genome has been sequenced,
the polymerase chain reaction (PCR) is used to
clone genes from libraries
• Gene-specific primers, complementary to the gene
of interest, are used to amplify the sequence
• It is also possible to modify genes by adding
desired base sequences to the DNA primers used
in amplification
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Epitope tagging
• Epitope tagging adds nucleotides to the amplified
sequence
• These encode a stretch of amino acids recognized
by commercially available antibodies
• When the amplified gene is expressed in cells, the
protein product can be detected by the antibody
(e.g., polyhistidine tagging, or His tagging)
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Genetic Engineering
• Genetic engineering involves the application of
recombinant DNA technology to practical
problems, especially in medicine and agriculture
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Genetic Engineering Can Produce
Valuable Proteins That Are Otherwise
Difficult to Obtain
• Among the first proteins to be produced by genetic
engineering was human insulin, required by
diabetics
• There are several ways to produce human insulin
from genetically engineered bacteria
• Other proteins produced this way are blood-clotting
factors, growth hormone, tissue plasminogen
activator, and other
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The Ti Plasmid Is a Useful Vector for
Introducing Foreign Genes into Plants
• Cloned genes can be transferred into plants by first
inserting them into Ti plasmid, which naturally
occurs in Agrobacterium tumefaciens
• A small part of the plasmid, the T DNA region,
usually inserts into the plant chromosomal DNA
• It causes uncontrolled growth of tissue called a
crown gall tumor
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Modification of the Ti plasmid for cloning
• In the laboratory, the sequences of the Ti plasmid
that cause tumor formation have been removed
• Inserting genes of interest into the modified
plasmids allows transfer of foreign genes into cells
• The gene is put into the plasmid, which is then put
into Agrobacterium cells
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Figure 20-31
© 2012 Pearson Education, Inc.
Producing transgenic plants
• Transformed Agrobacterium are used to infect plant
cells growing in culture
• These cells are used to generate plants containing
the foreign gene
• Such plants are said to be transgenic; a term to
describe any type of organism that carries one or
more genes from another organism (also called
GM, genetically modified)
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Genetic Modification Can Improve
the Traits of Food Crops
• Scientists have created many new GM crops
exhibiting a variety of new traits
– Plants can be made resistant to insect damage
by introducing a gene cloned from soil bacteria,
Bacillus thuringiensis (Bt); this gene produces a
protein very toxic to some insects
– “Golden rice” that has high-carotene content
has been produced to help address vitamin A
deficiency
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Concerns Have Been Raised About
the Safety and Environmental Risks
of GM Crops
• For consumers, the main focus has been on safety,
especially concerning possible allergic reactions
• However, a single gene inserted into a crop can be
easily assessed for safety hazards
• Environmental concerns have also been raised,
such as the possibility that plants containing the Bt
gene might be hazardous to beneficial insects
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Concerns about GM crops
• Despite legitimate concerns there has so far been
little evidence of significant environmental or
health risks
• GM crops have allowed for reduced use of
pesticides and may be beneficial in that regard
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Animals Can Be Genetically Modified
by Adding or Knocking Out Specific
Genetic Elements
• Techniques for genetically engineering animals
varies among different animals but often includes
microinjection of engineered DNA
• Palmiter and Brinster transferred the gene for
growth hormone into a fertilized mouse egg to
produce a transgenic mouse
• Proteins can be fused with GFP so that their
locations can be followed in living cells
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Figure 20A-1
© 2012 Pearson Education, Inc.
Practical applications of genetic
engineering in animals
• Genetic engineering can produce farm animals that
synthesize medically important proteins
• These can be produced, for instance, in the milk of
female mammals
• Engineered livestock are produced as a food source
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Removal of genes
• Removing a gene of interest or inactivation of it, is
referred to as “knock out”
• Homologous recombination can be used for this;
hundreds of stains of knockout mice have been
created, each defective in a single gene
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Production of knockout mice
• DNA is synthesized that is similar in base sequence
to the target gene and flanking sequences but with
two changes (1)
– An antibiotic resistance gene is inserted into the
target sequence
– DNA encoding the enzyme thymidine kinase is
attached to the end of the DNA; cells containing this
DNA will die if treated with an antiviral drug
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Figure 20-32
© 2012 Pearson Education, Inc.
Production of a knockout mouse
• The engineered DNA is introduced into embryonic
stem cells (2)
• In rare cases the DNA enters the nucleus and the
artificial DNA aligns with complementary
sequences flanking the target gene
• Homologous recombination replaces the target
gene with the engineered gene
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Selection of ES cells containing the
engineered gene
• If homologous recombination occurs, the
engineered gene confers antibiotic resistance to
the ES cells (3)
• In addition, the thymidine kinase gene is removed
from the engineered DNA and degraded; cells
treated with antiviral drugs will survive (4)
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Producing adult mice with the knocked
out gene
• ES cells identified using the double drug selection
are introduced into mouse embryos, which develop
into adult mice
• Some tissues in the mice have the inactivated
gene (5)
• Crossbreeding these animals eventually produces
strains of pure knockout mice (6)
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Gene Therapies Are Being Developed
for the Treatment of Human Diseases
• Gene transplantation techniques might be used to
repair defective genes in humans, referred to as
gene therapy
• A candidate for this is severe combined
immunodeficiency (SCID)
• The first person to be treated with gene therapy
was a girl with SCID caused by a defect in the
adenosine deaminase (ADA) gene
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Gene therapies
• The girl suffered from frequent, life-threatening
infections
• In 1990 she underwent treatments whereby a
cloned ADA gene was inserted into a virus, which
was used to infect T lymphocytes from her blood,
with the lymphocytes then injected into her
bloodstream
• She experienced improvement, but the effect
diminished over time
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Gene therapies—improvements
• In 2000, scientists reported a successful treatment
for SCID using a more efficient virus and better
conditions for culturing cells during gene transfer
• Immune function was restored to the children; but
later some developed leukemia, due to the virus
causing insertional mutagenesis of a normal gene
• The virus used was a retrovirus, whereas adenoassociated virus is less likely to inactivate host
genes
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Gene therapies—clotting factor
• Patients with hemophilia have deficiencies in
blood-clotting factors
• Hemophilia patients have been injected with
adeno-associated virus containing a gene coding
for the blood-clotting factor they require
• The cure was short lived, but promising
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