Chapter 17
Rearrangement of DNA
17.1 Introduction
17.2 The mating pathway is triggered by pheromone-receptor interactions
17.3 The mating response activates a G protein
17.4 Yeast can switch silent and active loci for mating type
17.5 The MAT locus codes for regulator proteins
17.6 Silent cassettes at HML and HMR are repressed
17.7 Unidirectional transposition is initiated by the recipient MAT locus
17.8 Regulation of HO expression
17.9 Trypanosomes switch the VSG frequently during infection
17.10 New VSG sequences are generated by gene switching
17.11 VSG genes have an unusual structure
17.12 The bacterial Ti plasmid causes crown gall disease in plants
17.13 T-DNA carries genes required for infection
17.14 Transfer of T-DNA resembles bacterial conjugation
17.15 Selection of amplified genomic sequences
17.16 Transfection introduces exogenous DNA into cells
17.17 Genes can be injected into animal eggs
17.18 ES cells can be incorporated into embryonic mice
17.19 Gene targeting allows genes to be replaced or knocked out
Amplification refers to the production of
additional copies of a chromosomal
sequence,found as intrachromosomal or
extrachromosomal DNA.
Transgenic animals are created by
introducing new DNA sequences into the
germline via addition to the egg.
17.1 Introduction
Amplification refers to the production of
additional copies of a chromosomal
sequence,found as intrachromosomal or
extrachromosomal DNA.
Transgenic animals are created by
introducing new DNA sequences into the
germline via addition to the egg.
17.1 Introduction
Figure 17.1 Mating type controls several activities.
17.2 The mating pathway is triggered by
signal transduction
Figure 17.2 The yeast life
cycle proceeds through
mating of MATa and
MATa haploids to give
heterozygous diploids
that sporulate to generate
haploid spores.
17.2 The mating
pathway is triggered
by signal transduction
Figure 17.3 Either a or a
factor/receptor interaction
triggers the activation of a
G protein,whose bg
subunits transduce the
signal to the next stage in
the pathway.
17.2 The mating
pathway is triggered
by signal transduction
Figure 17.4 The same mating
type response is triggered by
interaction of either pheromone
with its receptor,The signal is
transmitted through a series of
kinases to a transcription factor;
there may be branches to some
of the final functions.
17.2 The mating
pathway is triggered by
signal transduction
Figure 26.29 Homologous proteins are found in signal
transduction cascades in a wide variety of organisms.
17.2 The mating pathway is
triggered by signal transduction
Figure 17.5 Changes of
mating type occur when
silent cassettes replace active
cassettes of opposite
genotype; when
transpositions occur between
cassettes of the same type,
the mating type remains
unaltered.
17.3 Yeast can switch
silent and active loci
for mating type
Figure 17.6 Silent cassettes
have the same sequences as
the corresponding active
cassettes,except for the
absence of the extreme
flanking sequences in HMRa,
Only the Y region changes
between a and a types.
17.3 Yeast can switch
silent and active loci
for mating type
Figure 17.7 In diploids the a1 and a2 proteins cooperate to
repress haploid-specific functions,In a haploids,mating
functions are constitutive,In a haploids,the a2 protein
represses a mating functions,while a1 induces a mating
functions.
17.3 Yeast can switch silent and active loci for mating type
Figure 17.8
Combinations of
PRTF,a1,a1 and
a2 activate or
repress specific
groups of genes to
correspond with
the mating type of
the cell.
17.3 Yeast can switch silent and active loci for mating type
Figure 9.10 RNA polymerase
initially contacts the region
from -55 to +20,When sigma
dissociates,the core enzyme
contracts to -30; when the
enzyme moves a few base
pairs,it becomes more
compactly organized into the
general elongation complex.
9.4 Sigma factor
controls binding
to DNA
Figure 17.6 Silent
cassettes have the same
sequences as the
corresponding active
cassettes,except for the
absence of the extreme
flanking sequences in
HMRa,Only the Y
region changes between
a and a types.
17.4 Silent cassettes at HML and HMR are repressed
Figure 17.9 HO
endonuclease
cleaves MAT just
to the right of the
Y region,
generating sticky
ends with a base
overhang.
17.5 Unidirectional transposition is
initiated by the recipient MAT locus
Figure 17.10 Cassette
substitution is initiated by a
double-strand break in the
recipient (MAT) locus,and
may involve pairing on either
side of the Y region with the
donor (HMR or HML) locus.
17.5 Unidirectional
transposition is initiated
by the recipient MAT
locus
Figure 14.5 Recombination
is initiated by a double-
strand break,followed by
formation of single-
stranded 3 ends,one of
which migrates to a
homologous duplex.
9.4 Sigma factor controls
binding to DNA
Figure 17.11 Switching occurs only in mother cells; both daughter
cells have the new mating type,A daughter cell must pass through an
entire cycle before it becomes a mother cell that is able to switch again.
17.6 Regulation of HO expression
Figure 17.12 Three
regulator systems act
on transcription of the
HO gene,Transcription
occurs only when all
repression is lifted.
17.6 Regulation of
HO expression
Figure 17.13 A
trypanosome passes through
several morphological
forms when its life cycle
alternates between a tsetse
fly and mammalian host.
17.7 Trypanosomes
rearrange DNA to express
new surface antigens
Figure 17.14 The C-
terminus of VSG is
cleaved and covalently
linked to the membrane
through a glycolipid.
17.7
Trypanosomes
rearrange DNA
to express new
surface antigens
Figure 17.15 VSG
genes may be created
by duplicative transfer
from an internal or
telomeric basic copy
into an expression site,
or by activating a
telomeric copy that is
already present at a
potential expression
site.
17.7 Trypanosomes rearrange DNA to
express new surface antigens
Figure 17.16 Internal basic copies can be activated only by generating
a duplication of the gene at an expression-linked site
17.7 Trypanosomes rearrange DNA to
express new surface antigens
Figure 17.17 Telomeric basic copies can be activated in situ; the size of the
restriction fragment may change (slightly) when the telomere is extended.
17.7 Trypanosomes rearrange DNA
to express new surface antigens
Figure 17.18 The
expression-linked copy of a
VSG gene contains barren
regions on either side of the
transposed region,which
extends from ~1000 bp
upstream of the VSG coding
region to a site near the 3
terminus of the mRNA.
17.7 Trypanosomes
rearrange DNA to
express new surface
antigens
Figure 17.19 An
Agrobacterium carrying a Ti
plasmid of the nopaline type
induces a teratoma,in which
differentiated structures
develop,Photograph kindly
provided by Jeff Schell.
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.20 Ti plasmids carry genes involved
in both plant and bacterial functions.
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.21 T-DNA is
transferred from
Agrobacterium carrying a Ti
plasmid into a plant cell,
where it becomes integrated
into the nuclear genome and
expresses functions that
transform the host cell.
17.8 Interaction of Ti plasmid
DNA with the plant genome
Figure 17.22 Nopaline and octopine Ti plasmids
carry a variety of genes,including T-regions that
have overlapping functions
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.23 The vir
region of the Ti plasmid
has six loci that are
responsible for
transferring T-DNA to an
infected plant.
17.8 Interaction of Ti
plasmid DNA with the
plant genome
Figure 17.24
Acetosyringone
(4-acetyl-2,6-
dimethoxyphenol)
is produced by N,
tabacum upon
wounding,and
induces transfer of
T-DNA from
Agrobacterium.
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.25 The two-
component system of
VirA-VirG responds to
phenolic signals by
activating transcription
of target genes.
17.8 Interaction of Ti
plasmid DNA with the
plant genome
Figure 17.26 T-DNA has almost identical repeats of 25 bp at each end in
the Ti plasmid,The right repeat is necessary for transfer and integration
to a plant genome,T-DNA that is integrated in a plant genome has a
precise junction that retains 1-2 bp of the right repeat,but the left
junction varies and may be up to 100 bp short of the left repeat.
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.27 T-DNA is
generated by displacement
when DNA synthesis starts
at a nick made at the right
repeat,The reaction is
terminated by a nick at the
left repeat.
17.8 Interaction of Ti
plasmid DNA with the
plant genome
Figure 17.27 T-DNA is
generated by displacement
when DNA synthesis starts
at a nick made at the right
repeat,The reaction is
terminated by a nick at the
left repeat.
17.8 Interaction of Ti
plasmid DNA with the
plant genome
Amplification refers to the production
of additional copies of a chromosomal
sequence,found as intrachromosomal
or extrachromosomal DNA.
17.9 Selection of amplified genomic sequences
Figure 17.28 The dhfr gene
can be amplified to give
unstable copies that are
extrachromosomal (double
minutes) or stable
(chromosomal),
Extrachromosomal copies
arise at early times.
17.9 Selection of
amplified genomic
sequences
Figure 17.29 Amplified copies of the dhfr gene produce a
homogeneously staining region (HSR) in the chromosome,
Photograph kindly provided by Robert Schimke.
17.9 Selection of amplified genomic sequences
Figure 17.30 Amplified extrachromosomal dhfr genes
take the form of double-minute chromosomes,as seen
in the form of the small white dots,Photograph kindly
provided by Robert Schimke.
17.9 Selection of amplified genomic sequences
Figure 17.30 Amplified extrachromosomal dhfr genes
take the form of double-minute chromosomes,as seen
in the form of the small white dots,Photograph kindly
provided by Robert Schimke.
17.9 Selection of amplified genomic sequences
Transfection of eukaryotic cells is the
acquisition of new genetic markers by
incorporation of added DNA.
Transgenic animals are created by
introducing new DNA sequences into the
germline via addition to the egg.
17.10 Exogenous sequences can be introduced
into cells and animals by transfection
Figure 17.31 Transfection can introduce DNA
directly into the germ line of animals
17.10 Exogenous sequences can be introduced
into cells and animals by transfection
Figure 17.32 A
transgenic mouse
with an active rat
growth hormone
gene (left) is twice
the size of a normal
mouse (right),
Photograph kindly
provided by Ralph
Brinster.
17.10 Exogenous sequences can be introduced
into cells and animals by transfection
Figure 17.33 Hypogonadism of
the hpg mouse can be cured by
introducing a transgene that has
the wild-type sequence.
17.10 Exogenous
sequences can be
introduced into cells and
animals by transfection
Figure 17.34 ES cells
can be used to generate
mouse chimeras,which
breed true for the
transfected DNA when
the ES cell contributes
to the germ line.
17.10 Exogenous
sequences can be
introduced into cells
and animals by
transfection
Figure 17.35 A transgene
containing neo within an
exon and TK downstream
can be selected by
resistance to G418 and
loss of TK activity.
17.10 Exogenous
sequences can be
introduced into cells and
animals by transfection
Figure 17.36 Transgenic flies
that have a single,normally
expressed copy of a gene can be
obtained by injecting D,
melanogaster embryos with an
active P element plus foreign
DNA flanked by P element ends.
17.10 Exogenous sequences
can be introduced into cells
and animals by transfection
17.11 Summary
?Yeast mating type is determined by
whether the MAT locus carries the a or
sequence,
?Additional,silent copies of the mating-
type sequences are carried at the loci HML
and HMRa,
?Trypanosomes carry >1000 sequences
coding for varieties of the surface antigen,
?Agrobacteria induce tumor formation in wounded
plant cells,The wounded cells secrete phenolic
compounds that activate vir genes carried by the Ti
plasmid of the bacterium,
?Endogenous sequences may become amplified in
cultured cells,Exposure to methotrexate leads to the
accumulation of cells that have additional copies of
the dhfr gene,
?New sequences of DNA may be introduced into a
cultured cell by transfection or into an animal egg by
microinjection.
17.11 Summary
Rearrangement of DNA
17.1 Introduction
17.2 The mating pathway is triggered by pheromone-receptor interactions
17.3 The mating response activates a G protein
17.4 Yeast can switch silent and active loci for mating type
17.5 The MAT locus codes for regulator proteins
17.6 Silent cassettes at HML and HMR are repressed
17.7 Unidirectional transposition is initiated by the recipient MAT locus
17.8 Regulation of HO expression
17.9 Trypanosomes switch the VSG frequently during infection
17.10 New VSG sequences are generated by gene switching
17.11 VSG genes have an unusual structure
17.12 The bacterial Ti plasmid causes crown gall disease in plants
17.13 T-DNA carries genes required for infection
17.14 Transfer of T-DNA resembles bacterial conjugation
17.15 Selection of amplified genomic sequences
17.16 Transfection introduces exogenous DNA into cells
17.17 Genes can be injected into animal eggs
17.18 ES cells can be incorporated into embryonic mice
17.19 Gene targeting allows genes to be replaced or knocked out
Amplification refers to the production of
additional copies of a chromosomal
sequence,found as intrachromosomal or
extrachromosomal DNA.
Transgenic animals are created by
introducing new DNA sequences into the
germline via addition to the egg.
17.1 Introduction
Amplification refers to the production of
additional copies of a chromosomal
sequence,found as intrachromosomal or
extrachromosomal DNA.
Transgenic animals are created by
introducing new DNA sequences into the
germline via addition to the egg.
17.1 Introduction
Figure 17.1 Mating type controls several activities.
17.2 The mating pathway is triggered by
signal transduction
Figure 17.2 The yeast life
cycle proceeds through
mating of MATa and
MATa haploids to give
heterozygous diploids
that sporulate to generate
haploid spores.
17.2 The mating
pathway is triggered
by signal transduction
Figure 17.3 Either a or a
factor/receptor interaction
triggers the activation of a
G protein,whose bg
subunits transduce the
signal to the next stage in
the pathway.
17.2 The mating
pathway is triggered
by signal transduction
Figure 17.4 The same mating
type response is triggered by
interaction of either pheromone
with its receptor,The signal is
transmitted through a series of
kinases to a transcription factor;
there may be branches to some
of the final functions.
17.2 The mating
pathway is triggered by
signal transduction
Figure 26.29 Homologous proteins are found in signal
transduction cascades in a wide variety of organisms.
17.2 The mating pathway is
triggered by signal transduction
Figure 17.5 Changes of
mating type occur when
silent cassettes replace active
cassettes of opposite
genotype; when
transpositions occur between
cassettes of the same type,
the mating type remains
unaltered.
17.3 Yeast can switch
silent and active loci
for mating type
Figure 17.6 Silent cassettes
have the same sequences as
the corresponding active
cassettes,except for the
absence of the extreme
flanking sequences in HMRa,
Only the Y region changes
between a and a types.
17.3 Yeast can switch
silent and active loci
for mating type
Figure 17.7 In diploids the a1 and a2 proteins cooperate to
repress haploid-specific functions,In a haploids,mating
functions are constitutive,In a haploids,the a2 protein
represses a mating functions,while a1 induces a mating
functions.
17.3 Yeast can switch silent and active loci for mating type
Figure 17.8
Combinations of
PRTF,a1,a1 and
a2 activate or
repress specific
groups of genes to
correspond with
the mating type of
the cell.
17.3 Yeast can switch silent and active loci for mating type
Figure 9.10 RNA polymerase
initially contacts the region
from -55 to +20,When sigma
dissociates,the core enzyme
contracts to -30; when the
enzyme moves a few base
pairs,it becomes more
compactly organized into the
general elongation complex.
9.4 Sigma factor
controls binding
to DNA
Figure 17.6 Silent
cassettes have the same
sequences as the
corresponding active
cassettes,except for the
absence of the extreme
flanking sequences in
HMRa,Only the Y
region changes between
a and a types.
17.4 Silent cassettes at HML and HMR are repressed
Figure 17.9 HO
endonuclease
cleaves MAT just
to the right of the
Y region,
generating sticky
ends with a base
overhang.
17.5 Unidirectional transposition is
initiated by the recipient MAT locus
Figure 17.10 Cassette
substitution is initiated by a
double-strand break in the
recipient (MAT) locus,and
may involve pairing on either
side of the Y region with the
donor (HMR or HML) locus.
17.5 Unidirectional
transposition is initiated
by the recipient MAT
locus
Figure 14.5 Recombination
is initiated by a double-
strand break,followed by
formation of single-
stranded 3 ends,one of
which migrates to a
homologous duplex.
9.4 Sigma factor controls
binding to DNA
Figure 17.11 Switching occurs only in mother cells; both daughter
cells have the new mating type,A daughter cell must pass through an
entire cycle before it becomes a mother cell that is able to switch again.
17.6 Regulation of HO expression
Figure 17.12 Three
regulator systems act
on transcription of the
HO gene,Transcription
occurs only when all
repression is lifted.
17.6 Regulation of
HO expression
Figure 17.13 A
trypanosome passes through
several morphological
forms when its life cycle
alternates between a tsetse
fly and mammalian host.
17.7 Trypanosomes
rearrange DNA to express
new surface antigens
Figure 17.14 The C-
terminus of VSG is
cleaved and covalently
linked to the membrane
through a glycolipid.
17.7
Trypanosomes
rearrange DNA
to express new
surface antigens
Figure 17.15 VSG
genes may be created
by duplicative transfer
from an internal or
telomeric basic copy
into an expression site,
or by activating a
telomeric copy that is
already present at a
potential expression
site.
17.7 Trypanosomes rearrange DNA to
express new surface antigens
Figure 17.16 Internal basic copies can be activated only by generating
a duplication of the gene at an expression-linked site
17.7 Trypanosomes rearrange DNA to
express new surface antigens
Figure 17.17 Telomeric basic copies can be activated in situ; the size of the
restriction fragment may change (slightly) when the telomere is extended.
17.7 Trypanosomes rearrange DNA
to express new surface antigens
Figure 17.18 The
expression-linked copy of a
VSG gene contains barren
regions on either side of the
transposed region,which
extends from ~1000 bp
upstream of the VSG coding
region to a site near the 3
terminus of the mRNA.
17.7 Trypanosomes
rearrange DNA to
express new surface
antigens
Figure 17.19 An
Agrobacterium carrying a Ti
plasmid of the nopaline type
induces a teratoma,in which
differentiated structures
develop,Photograph kindly
provided by Jeff Schell.
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.20 Ti plasmids carry genes involved
in both plant and bacterial functions.
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.21 T-DNA is
transferred from
Agrobacterium carrying a Ti
plasmid into a plant cell,
where it becomes integrated
into the nuclear genome and
expresses functions that
transform the host cell.
17.8 Interaction of Ti plasmid
DNA with the plant genome
Figure 17.22 Nopaline and octopine Ti plasmids
carry a variety of genes,including T-regions that
have overlapping functions
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.23 The vir
region of the Ti plasmid
has six loci that are
responsible for
transferring T-DNA to an
infected plant.
17.8 Interaction of Ti
plasmid DNA with the
plant genome
Figure 17.24
Acetosyringone
(4-acetyl-2,6-
dimethoxyphenol)
is produced by N,
tabacum upon
wounding,and
induces transfer of
T-DNA from
Agrobacterium.
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.25 The two-
component system of
VirA-VirG responds to
phenolic signals by
activating transcription
of target genes.
17.8 Interaction of Ti
plasmid DNA with the
plant genome
Figure 17.26 T-DNA has almost identical repeats of 25 bp at each end in
the Ti plasmid,The right repeat is necessary for transfer and integration
to a plant genome,T-DNA that is integrated in a plant genome has a
precise junction that retains 1-2 bp of the right repeat,but the left
junction varies and may be up to 100 bp short of the left repeat.
17.8 Interaction of Ti plasmid DNA with the plant genome
Figure 17.27 T-DNA is
generated by displacement
when DNA synthesis starts
at a nick made at the right
repeat,The reaction is
terminated by a nick at the
left repeat.
17.8 Interaction of Ti
plasmid DNA with the
plant genome
Figure 17.27 T-DNA is
generated by displacement
when DNA synthesis starts
at a nick made at the right
repeat,The reaction is
terminated by a nick at the
left repeat.
17.8 Interaction of Ti
plasmid DNA with the
plant genome
Amplification refers to the production
of additional copies of a chromosomal
sequence,found as intrachromosomal
or extrachromosomal DNA.
17.9 Selection of amplified genomic sequences
Figure 17.28 The dhfr gene
can be amplified to give
unstable copies that are
extrachromosomal (double
minutes) or stable
(chromosomal),
Extrachromosomal copies
arise at early times.
17.9 Selection of
amplified genomic
sequences
Figure 17.29 Amplified copies of the dhfr gene produce a
homogeneously staining region (HSR) in the chromosome,
Photograph kindly provided by Robert Schimke.
17.9 Selection of amplified genomic sequences
Figure 17.30 Amplified extrachromosomal dhfr genes
take the form of double-minute chromosomes,as seen
in the form of the small white dots,Photograph kindly
provided by Robert Schimke.
17.9 Selection of amplified genomic sequences
Figure 17.30 Amplified extrachromosomal dhfr genes
take the form of double-minute chromosomes,as seen
in the form of the small white dots,Photograph kindly
provided by Robert Schimke.
17.9 Selection of amplified genomic sequences
Transfection of eukaryotic cells is the
acquisition of new genetic markers by
incorporation of added DNA.
Transgenic animals are created by
introducing new DNA sequences into the
germline via addition to the egg.
17.10 Exogenous sequences can be introduced
into cells and animals by transfection
Figure 17.31 Transfection can introduce DNA
directly into the germ line of animals
17.10 Exogenous sequences can be introduced
into cells and animals by transfection
Figure 17.32 A
transgenic mouse
with an active rat
growth hormone
gene (left) is twice
the size of a normal
mouse (right),
Photograph kindly
provided by Ralph
Brinster.
17.10 Exogenous sequences can be introduced
into cells and animals by transfection
Figure 17.33 Hypogonadism of
the hpg mouse can be cured by
introducing a transgene that has
the wild-type sequence.
17.10 Exogenous
sequences can be
introduced into cells and
animals by transfection
Figure 17.34 ES cells
can be used to generate
mouse chimeras,which
breed true for the
transfected DNA when
the ES cell contributes
to the germ line.
17.10 Exogenous
sequences can be
introduced into cells
and animals by
transfection
Figure 17.35 A transgene
containing neo within an
exon and TK downstream
can be selected by
resistance to G418 and
loss of TK activity.
17.10 Exogenous
sequences can be
introduced into cells and
animals by transfection
Figure 17.36 Transgenic flies
that have a single,normally
expressed copy of a gene can be
obtained by injecting D,
melanogaster embryos with an
active P element plus foreign
DNA flanked by P element ends.
17.10 Exogenous sequences
can be introduced into cells
and animals by transfection
17.11 Summary
?Yeast mating type is determined by
whether the MAT locus carries the a or
sequence,
?Additional,silent copies of the mating-
type sequences are carried at the loci HML
and HMRa,
?Trypanosomes carry >1000 sequences
coding for varieties of the surface antigen,
?Agrobacteria induce tumor formation in wounded
plant cells,The wounded cells secrete phenolic
compounds that activate vir genes carried by the Ti
plasmid of the bacterium,
?Endogenous sequences may become amplified in
cultured cells,Exposure to methotrexate leads to the
accumulation of cells that have additional copies of
the dhfr gene,
?New sequences of DNA may be introduced into a
cultured cell by transfection or into an animal egg by
microinjection.
17.11 Summary
