Rearrangement of DNA

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Chapter 17

• Rearrangement of DNA

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17.1 Introduction 17.2 The mating pathway is
triggered by pheromone-receptor interactions17.3
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
locus17.8 Regulation of HO expression 17.9
Trypanosomes switch the VSG frequently during
infection17.10 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 plants17.13 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
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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
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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
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Figure 17.1 Mating type controls several
activities.
17.2 The mating pathway is triggered by signal
transduction
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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
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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
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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
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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
10
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
11
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
12
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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Figure 17.24 Acetosyringone (4-acetyl-2,6-dimethox
yphenol) 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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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 gt1000 sequences coding for
  varieties of the surface antigen.

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17.11 Summary

• 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.