Chapter 4 Molecular Cloning Methods

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Chapter 4 Molecular Cloning Methods
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Introduction The significance of gene cloning

• To elucidate the structure and function of genes.
• i.e. investigating hGH gene
• hGH gene lt10-6 of human genome
• Problem 1 need kilograms of human
• genome DNA for 1µg hGH gene
• Problem 2 how to separate the gene from the
• rest DNA

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4.1 Gene Cloning

• The procedure in a gene cloning experiment is
• To place a foreign gene into a bacterial cell
• To grow a clone of those modified bacteria.
• The principle factors for gene cloning
  experiment
• Restriction endonucleases
• Vectors
• Specific probe

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• The Role of Restriction Endonucleases
• Vectors
• Plasmids as Vectors
• Phages as Vectors
• ? Phage Vectors
• Cosmids
• M13 phage vectors
• Phagemids
• Eukaryotic Vectors
• Identifying a Specific Clone with a Specific
  Probe
• Polynucleotide Probes

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4.1.1 The Role of Restriction Endonucleases

• Restriction restrict the host range of the
  virus
• Endonucleases cut at sites within the foreign
  DNA
• How to name the first 3 letters of the Latin
  name of the microorganism the strain
  designation Roman numeral

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The main advantage of restriction enzyme is there
ability to cut a DNA reproducibly in the same
place this is the basis of many techniques used
to analyze genes. Many restriction enzymes
make staggered cut in the two DNA strands,
leaving a sticky ends, that can base-pair
together briefly.Enzymes that recognize
identical sequences are called isoschizomers.
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Restriction- modification systemR-M system

• Almost all restriction nucleases are paired with
  methylases that recognize and methylate the same
  DNA sites

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Figure 4.1 Maintaining restriction endonuclease
resistance after DNA replication We begin with
an EcoRI site that is methylated (red) on both
strands. After replication, the parental strand
of each daughter DNA duplex remains methylated,
but the newly made strand of each duplex has not
been methylated yet. The one methylated strand in
these hemimethylated DNAs is enough to protect
both strands against cleavage by EcoRI. Soon, the
methylase recognizes the unmethylated strand in
each EcoRI site and methylates it, regenerating
the fully methylated DNA.
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Figure 4.2 The first cloning experiment
involving a recombinant DNA assembled in vitro.
Boyer and Cohen cut two plasmids, pSC101 and
RSF1010, with the same restriction endonuclease,
EcoRI. This gave the twolinear DNAs the same
stickyends, which were then linked in vitro using
DNA ligase. The investigators reintroduced the
recombinant DNA into E. coli cells by
transformation and selected clones that were
resistant to both tetracycline and streptomycin.
These clones were therefore harboring the
recombinant plasmid.
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Summary
Restriction endonucleases recognize
specific sequences in DNA molecules and make cuts
in both strands. This allows very specific
cutting of DNAs. Also, because the cuts in the
two strands are frequently staggered, restriction
enzymes can create sticky ends that help link
together two DNAs to form a recombinant DNA in
vitro.
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4.1.2 Vectors

• Vectors serve as carriers to allow replication of
  recombinant DNAs.
• Origin of replication
• Multiple cloning site(MCS)
• Selection gene
• Plasmids pBR322 pUC
• Phages ?phage cosmids M13
• Phagemids

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Plasmids as Vectors
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Summary The first generations of plasmid
cloning vectors were pBR322 and the pUC plasmids.
The former has two antibiotic resistance genes
and a variety of unique restriction sites into
which one can introduce foreign DNA. Most of
these sites interrupt one of the antibiotic
resistance genes, making screening
straightforward. Screening is even easier with
the pUC plasmids. These have an ampicillin
resistance gene and a multiple cloning site that
interrupts a partial ß-galactosidase gene. One
screens for ampicillin-resistant clones that do
not make active ß-galactosidase and therefore do
not turn the indicator, X-gal, blue. The multiple
cloning site also makes it convenient to carry
out directional cloning into two different
restriction sites.
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Figure 4.3 The plasmid pBR322, showing the
locations of 11 unique restriction sites that can
be used to insert foreign DNA The locations
of the two antibiotic resistance genes (Ampr
ampicillin resistance Tetr tetracycline
resistance) and the origin of replication (ori )
are also shown. Numbers refer to kilobase pairs
(kb) from the EcoRI site.
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Figure 4.4 Cloning foreign DNA using the PstI
site of pBR322. We cut both the plasmid and
the insert (yellow) with PstI, then join them
through these sticky ends with DNA ligase. Next,
we transform bacteria with the recombinant DNA
and screen for tetracycline-resistant,
ampicillin-sensitive cells. The recombinant
plasmid no longer confers ampicillin resistance
because the foreign DNA interrupts that
resistance gene (blue).
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Figure 4.5 Screening bacteria by replica
plating. (a) The replica plating process. We
touch a velvet-covered circular tool to the
surface of the first dish containing colonies of
bacteria. Cells from each of these colonies stick
to the velvet and can be transferred to the
replica plate in the same positions relative to
each other. (b) Screening for inserts in the
pBR322 ampicillin resistance gene by replica
plating. The original plate contains
tetracycline, so all colonies containing pBR322
will grow. The replica plate contains ampicillin,
so colonies bearing pBR322 with inserts in the
ampicillin resistance gene will not grow (these
colonies are depicted by dotted circles). The
corresponding colonies from the original plate
can then be picked.
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pUC
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lacZ coding for the amino terminalportion of
the enzyme ß galactosidease. Host E.coli strain
carry a gene fragment that codes the carboxyl
potion of ß galactosidease When X-gal cleaved
by ß galactosidease, it releases galactose plus
an indigo dye that stains the bacterial colony
blue.
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Figure 4.7 Joining of vector to insert. (a)
Mechanism of DNA ligase. Step 1 DNA ligase
reacts with an AMP donoreither ATP or
NAD(nicotinamide adenine dinucleotide), depending
on the type of ligase. This produces an activated
enzyme (ligase-AMP). Step 2 The activated enzyme
donates a phosphate to the free 5-phosphate at
the nick in the lower strand of the DNA duplex,
creating a high-energy diphosphate group on one
side of the nick. Step 3 With energy provided by
cleavage of the diphosphate, a new phosphodiester
bond is created, sealing the nick in the DNA.
This reaction can also occur in both DNA strands
at once, so two independent DNAs can be joined
together by DNA ligase.
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Figure 4.7 Joining of vector to insert.
(b)Alkaline phosphatase prevents vector
re-ligation. Step 1 We cut the vector(blue,
top left) with BamHI. This produces sticky ends
with 5-phosphates(red). Step 2 We remove the
phosphates with alkaline phosphatase, making it
impossible for the vector to re-ligate with
itself. Step 3 We also cut the insert(yellow,
upper right) with BamHI, producing sticky ends
with phosphates that we do not remove. Step 4
Finally, we ligate the vector and insert
together. The phosphates on the insert allow two
phosphodiester bonds to form(red), but leave two
unformed bonds, or nicks, These will be completed
once the DNA is in the transformed bacterial
cell.
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Phages as vectors
Natural advantages over plasmid They infect
cells much more efficiently than plasmids
transform cells, so the yield of clones with
phage vectors is usually higher.
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Summary Two kinds of phages have been
especially popular as cloning vectors. The first
of these is ?, from which certain nonessential
genes have been removed to make room for inserts.
Some of these engineered phages can accommodate
inserts up to 20 kb, which makes them useful for
building genomic libraries, in which it is
important to have large pieces of genomic DNA in
each clone. Cosmids can accept even larger
insertsup to 50 kbmaking them a favorite choice
for genomic libraries. The second major class of
phage vector is composed of the M13 phages. These
vector have the convenience of a multiple cloning
site and the further advantage of producing
single-stranded recombinant DNA, which can be
used for DNA sequencing and for site-direct
mutagenesis. Plasmids called phagemids have also
been engineered to produce single-stranded DNA in
the presence of helper phages.
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Figure 4.8 Cloning in Charon 4. (a) Forming the
recombinant DNA. We cut the vector (yellow) with
EcoRI to remove the stuffer fragment and save the
arms. Next, we ligate partially digested insert
DNA (red) to the arms. (b) Packaging and
cloning the recombinant DNA. We mix the
recombinant DNA from (a) with an in vitro
packaging extract that contains ? phage head and
tail components and all other factors needed to
package the recombinant DNA into functional phage
particles. Finally, we plate these particles on
E.coli and collect the plaques that form.
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Figure 4.9 Selection of positive genomic clones
by plaque hybridization. First, we touch a
nitrocellulose ot similar filter to the surface
of the dish containing the Charon 4 plaques from
Figure 4.8. Phage DNA released naturally from
each plaque will stick to the filter. Next, we
denature the DNA with alkali and hybridize the
filter to a labeled probe for the gene we are
studying, then use X-ray film to reveal the
position of the label. Cloned DNA from one plaque
near the center of the filter has hybridized, as
shown by the dark spot on the film.
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Cosmids Behave both as plasmids and as
phages Contain the cos sites of ? and plasmid
origin of replication Have room for 40-50 kb
inserts.
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M13 phage vectors ß galactosidease gene
fragment pUC family MCS Single stranded DNA genome
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Figure 4.10 Obtaining single-stranded DNA by
cloning in M13 phage. Foreign DNA (red), cut
with HindIII, is inserted into the HindIII site
of the double-stranded phage DNA. The resulting
recombinant DNA is used to transform E.coli
cells, whereupon the DNA replicates by a rolling
circle mechanism, producing many single-stranded
product DNAs. The product DNAs are called
positive () strands, by convention. The template
DNA is therefore the negative (-) strand.
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Phagemides Single-stranded Both phage and
plasmid characteristics Help phage Two RNA
polymerase promoters (T7and T3)
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Summary
Two kinds of phages have been especially
popular as cloning vectors. The first of these is
?, from which certain nonessential genes have
been removed to make room for inserts. Some of
these engineered phages can accommodate inserts
up to 20 kb, which makes them useful for building
genomic libraries, in which it is important to
have large pieces of genomic DNA in each clone.
Cosmids can accept even larger insertsup to 50
kbmaking them a favorite choice for genomic
libraries. The second major class of phage vector
is composed of the M13 phages. These vector have
the convenience of a multiple cloning site and
the further advantage of producing
single-stranded recombinant DNA, which can be
used for DNA sequencing and for site-direct
mutagenesis. Plasmids called phagemids have also
been engineered to produce single-stranded DNA in
the presence of helper phages.
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4.1.3 Identifying a Specific Clone with a
Specific Probe

• Polynucleotide Probes
• High stringency
• Low stringency

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Summary

• Specific clones can be identified using
  polynucleotide probes that bind to the gene
  itself. Knowing the amino acid sequence of a gene
  product, one can design a set of oligonucleotides
  that encode part of this amino acid sequence.
  This can be one of the quickest and most accurate
  means of identifying a particular clone.

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4.2 The Polymerase Chain Reaction (PCR)

• PCR amplifies a region of DNA between
  two predetermined sites. Oligo-nucleotides
  complementary to these sites serve as primers for
  synthesis of copies of the DNA between the sites.
  Each cycle of PCR double the number of copies of
  the amplified DNA until a large quantity has been
  made.

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Figure 4.12 Amplifying DNA by the polymerase
chain reaction. Cycle 1 Start with a
DNA duplex (top) and heat it to separate its two
strands (red and blue). Then add short,
single-stranded DNA primers (purple and yellow)
complementary to sequences on either side of the
region (X) to be amplified. The primers hybridize
to the appropriate sites on the separated DNA
strands now a special heat-stable DNA polymerase
uses these primers to start synthesis of
complementary DNA strands. The arrows represent
newly made DNA, in which replication has stopped
at the tip of the arrowhead. At the end of cycle
1, two DNA duplexes are present, including the
region to be amplified, whereas we started with
only one. The 5?3 polarities of all DNA strands
and primers are indicated throughout cycle 1. The
same principles apply in cycle 2. Cycle 2 Repeat
the process, heating to separate DNA strands,
cooling to allow annealing with primers, and
letting the heat-stable DNA polymerase make more
DNA. Now each of the four DNA strands, including
the two newly made ones, can serve as templates
for complementary DNA synthesis. The result is
four DNA duplexes that have the region to be
amplified. Notice that each cycle doubles the
number of molecules of DNA because the products
of each cycle join the parental molecules in
serving as templates for next cycle. This
exponential increase yields 8 molecules in the
next cycle and 16 in the cycle after that. This
process obviously leads to very high numbers in
only a short time.
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4.2.1 cDNA Cloning

• Nick translation
• Reverse transcriptase
• RNase H
• Terminal transferase

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Figure 4.13 Making a cDNA library. This
figure focuses on cloning a single cDNA , but the
method can be applied to a mixture of mRNAs and
produce a library of corresponding cDNAs. (a) Use
oligo(dT) as a primer and reverse transcriptase
tocopy the mRNA (blue), producing a cDNA (red)
that is hybridized to the mRNA template. (b) Use
RNase H to partially digest the mRNA, yielding a
set of RNA primers base-paired to the
first-strand cDNA. (c) Use E.coli DNA polymerase
I under nick translation conditions to build
second-strand cDNAs on the RNA primers. (d) The
second-strand cDNA growing from the leftmost
primer (blue) has been extended all the way to
the 3-end of the oligo(dA) corresponding to the
oligo(dT) primer on the first-strand cDNA. (e) To
give the double-stranded cDNA sticky ends, add
oligo(dC) with terminal transferase. (f) Anneal
the oligo(dC) ends of the cDNA to complementary
oligo(dG) ends of a suitable vector (black). The
recombinant DNA can then be used to transform
bacterial cells. Enzymes in these cells remove
remaining nicks and replace any remaining RNA
with DNA.
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Figure 4.15 Using RT-PCR to clone a single cDNA.
(a) Use a reverse primer (red) with a HindIII
site (yellow) at its 5-end to start first-strand
cDNA synthesis, with reverse transcriptase to
catalyze the reaction. (b) Denature the mRNA-cDNA
hybrid and anneal a forward primer (red) with a
BamHI site (green) at its 5-end. (c) This
forward primer initiates second-strand cDNA
synthesis, with DNA polymerase catalyzing the
reaction. (d) Continue PCR with the same two
primers to amplify the double-stranded cDNA. (e)
Cut the cDNA with BamHI and HindIII to generate
sticky ends. (f) Ligate the cDNA to the BamHI and
HindIII sites of a suitable vector (purple).
Finally, transform cells with the recombinant
cDNA to produce a clone.
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Figure 4.16 RACE procedure to fill in the 5-end
of a cDNA. (a) Hybridize an incomplete cDNA
(red), or an oligonucleotide segment of a cDNA to
mRNA (green), and use reverse transcriptase to
extend the cDNA to the 5-end of the mRNA. (b)
Use terminal transferase and dCTP to add C
residues to the 3end of the extended cDNA also,
use RNase H to degrade the mRNA. (c) Use an
oligo(dG) primer and DNA polymerase to synthesize
a second strand of cDNA (blue). (d) Perform PCR
with oligo(dG) as the forward primer and an
oligonucleotide that hybridizes to the 3-end of
the cDNA as the reverse primer. (e)The product is
a cDNA that has been extended to the 5-end of
the mRNA. A similar procedure (3-RACE) can be
used to extend the cDNA in the 3-direction. In
that case, there is no need to tail the 3-end of
the cDNA with terminal transferase because the
mRNA already contains poly(A) thus, the reverse
primer would be oligo(dT).
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Summary To make a cDNA library, we can
synthesize cDNAs one strand at a time, using
mRNAs from a cell as templates for the first
strands and these first strands as temletes for
the second strands. Reverse trnscriptase
generates the first strands and E.coli DNA
polymerase I generates the second strands. We can
endow the double stranded cDNAs with
oligonucleotide tails that base-par with
complementary tails on a cloning vector. We can
then use these recombinant DNAs to transform
bacteria. We can use RT-PCR to generate a cDNA
from a single type of mRNA, but we must know the
sequence of the mRNA in order to design the
primers for the PCR step. If we put restriction
sites on the PCR primers, we place these sites at
the ends of the cDNA,so it is easy to ligate the
cDNA into a vector. We can detect particular
clones by colony hybridazation with redioactive
DNA probes,or with antibodies if an expression
vector such as ?gt11 is used.
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4.3 Methods of Expressing Cloned Genes

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4.3.1 Expression Vectors

• Expression vectors with strong promoters
• Inducible Expression Vectors
• Expression vectors produce fusion proteins
• Eukaryotic expression vectors

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Figure 4.17 Producing a fusion protein by cloning
in a pUC plasmid. Insert foreign DNA
(yellow) into the multiple cloning site (MCS)
transcription from the lac promoter (purple)
gives a hybrid mRNA beginning with a few lacZ
codons, changing to insert sequence, then back to
lacZ (red). This mRNA is translated to a fusion
protein containing a few ß-galactosidase amino
acids for the remainder ofthe protein. Because
the insert contains a translation stop codon, the
remaining lacZ codons are not translated.
Figure 4.17
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Figure 4.18 Using a PBAD vector. The
green fluorescent protein (GFP) gene was cloned
into a vector under control of the PBAD promoter
and promoter activity was induced with increasing
concentrations of arabinose. GFP production was
monitored by electrophoresing extracts from cells
induced with the arabinose concentrations given
at top, blotting the proteins to a membrane, and
detecting GFP with an anti-GFP antibody .
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Summary Expression vectors are designed to
yield the protein product of a cloned gene,
usually in the greatest amount possible. To
optimize expression, these vectors provide strong
bacterial promoters and bacterial ribosome
binding sites that would be missing on cloned
eukaryotic genes. Most cloning vectors are
inducible, to avoid premature overproduction of a
foreign product that could poison the bacterial
host cells.
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Figure 4.19 Using an oligohistidine expression
vector. (a) Map of a generic oligohistidine
vector. Just after the ATG initiation
codon (green) lies a coding region (red) encoding
six histidine in a row (His)6. This is followed
by a region (orange) encoding a recognition site
for the proteolytic enzyme enterokinase (EK).
Finally, the vector has a multiple cloning site
(MCS, blue). Usually, the vector comes in three
forms with the MCS sites in each of the three
reading frames. One can select the vector that
puts the gene in the right reading frame relative
to the oligohistidine.
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Figure 4.19 Using an oligohistidine expression
vector. (b) Using the vector. 1. Insert the gene
of interest (yellow) into the vector in frame
with the oligohistidine coding region (red) and
transform bacterial cells with the recombinant
vector. The cells produce the fusion protein (red
and yellow), along with other, bacterial proteins
(green). 2. Lyse the cells, releasing the
mixture of proteins. 3. Pour the cell lysate
through a nickel affinity chromatography column,
which binds the fusion protein but not the other
proteins. 4. Release the fusion protein from the
column with histidine or with imidazole, a
histidine analogue, which competes with the
oligohistidine for binding to the nickel. 5.
Cleave the fusion protein with enterokinase. 6.
Pass the cleaved protein through the nickel
column once more to separate the oligehistidine
from the desired protein.
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Summary Expression vectors frequently
produce fusion proteins, with one part of the
protein coming from coding sequences in the
vector and the other part from sequences in the
cloned gene itself. Many fusion proteins have the
great advantage of being simple to isolate by
affinity chromatography. The ?gt11 vector
produces fusion proteins that can be detected in
plaques with a specific antiserum.
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Figure 4.20 Forming a fusion protein in ?gt11.
The gene to be expressed (green) is inserted
into the EcoRI site near the end of the lacZ
coding region (red) just upstream of the
transcription terminator. Thus, upon induction of
the lacZ gene by IPTG, a fused mRNA results,
containing the inserted coding region just
downstream of that of ß-galactosidase. This mRNA
is translated by the host cell to yield a fusion
protein.
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Figure 4.21 Detecting positive?gt11 clones by
antibody screening. A filter is used to
blot proteins from phage plaques on a Petri dish.
One of the clones (red) has produced a plaque
containing a fusion protein including
ß-galactosidase and a part of the protein of
interest. The filter with its blotted proteins is
incubated with an antibody directed against our
protein of interest, then with labeled
Staphylococcus protein A, which binds
specifically to antibodies. It will therefore
bind only to the antibody-antigen complexes at
the spot corresponding to our positive clone. A
dark spot on the film placed in contact with the
filter reveals the location of our positive
clone.
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Figure 4.22 Expressing a gene in a baculovirus.
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Figure 4.22 Expressing a gene in a baculovirus.
First, insert the gene to be expressed
(red), into a baculovirus transfer vector. In
this case, the vector contains the powerful
polyhedrin promoter (Polh), flanked bythe DNA
sequences (yellow) that normally surround the
polyhedrin gene, including a gene (green) that is
essential for virus replication, the polyhedrin
coding region itself is missing from this
transfer vector. Just downstream of the promoter
is a BamHI restriction site, which can be used to
open up the vector (step a) so it can accept the
foreign gene (red) by ligation (step b). In step
c, mix the recombinant transfer vector with
linear viral DNA that has been cut so as to
remove the essential gene. Transfect insect cells
with the two DNAs together. This process is known
as co-transfection. The two DNAs are not drawn to
scale, the viral DNA is actually almost 15 times
the size of the vector. Inside the cell, the two
DNAs recombine by a double crossover that inserts
the gene to be expressed, along with the
essential gene, into the viral DNA. The result is
a recombinant virus DNA that has the gene of
interest under the control of the polyhedrin
promoter. Next, infect cells with the recombinant
virus. Finally, in step d and e, infect cells
with the recombinant virus and collect the
protein product these cells make. Notice that the
original viral DNA is linear and it is missing
the essential gene , so it cannot infect cells
(f). This lack of infectivity selects
automatically for recombinant viruses they are
the only ones that can infect cells.
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Summary Foreign genes can be expressed in
eukaryotic cells, and these eukaryotic systems
have some advantages over their prokaryotic
counterparts for producing eukaryotic proteins.
Two of the most important advantages are (1)
Eukaryotic proteins made in eukaryotic cells tend
to be folded properly, so they are soluble,
rather than aggregated into insoluble inclusion
bodies. (2) Eukaryotic proteins made in
eukaryotic cells are modified (phosphorylated,
glycosylated, etc.) in a eukaryotic manner.
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4.3.2 Other Eukaryotic Vectors

• Yeast Artificial chromosomes (YACs)
• Using the Ti plasmid to transfer genes to plants

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• Essential components of YAC vectors
• Centromers (CEN), telomeres (TEL) and autonomous
  replicating sequence (ARS) for proliferation in
  the host cell.
• ampr for selective amplification and markers such
  as TRP1 and URA3 for identifying cells containing
  the YAC vector.
• Recognition sites of restriction enzymes (e.g.,
  EcoRI and BamHI)

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BAC vectors

• Bacterial artificial chromosomes are
  based on the F factor of E. coli and can be used
  to clone up to 350 kb of genomic DNA in a
  conveniently handled E. coli host. They are a
  morre stable and easier to use alternative to YAC.

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Using the Ti Plasmid to Transfer Gees to Plants
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Nopaline and octopine Ti plasmids carry
a variety of genes, including T-regions that have
overlapping functions
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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.
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Figure 4.24 Crown gall tumors. (a) Formation of a
crown gall. 1. Agrobacterium cells enter a wound
in the plant, usually at the crown, or the
junction of root an stem. 2. The Agrobacterium
contains a Ti plasmid in addition to the much
larger bacterial chromosome. The Ti plasmid has a
segment (the T-DNA, red) that promotes tumor
formation in infected plants. 3. The bacterium
contributes its Ti plasmid to the plant cell, and
the T-DNA from the Ti plasmid into grates into
the plants chromosomal DNA. 4. The genes in the
T-DNA direct the formation of a crown gall, which
nourishes the invading bacteria.
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Figure 4.24 Crown gall tumors. (b) Photograph of
a crown gall tumor genetated by cutting off the
top of a tobacco plant and inoculating with
Agrobacterium. This crown gall tumor is a
teratoma, which generates normal as well as
tumorous tissues springing from the tumor.
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Figure 4.25 Using a T-DNA plasmid to introduce a
gene into tobacco plant. (a) A plasmid is
formed with a foreign gene (red) under the
control of the mannopine synthetase promoter
(blue). This plasmid is used to transform
Agrobacterium cells. (b) The transformed
bacterial cells divide repeatedly. (c) A disk of
tobacco leaf tissue is removed and incubated in
nutrient medium, along with the transformed
Agrobacterium cells. These cells infect the
tobacco tissue, transferring the plasmid bearing
the cloned foreign gene. (d) The disk of tobacco
tissue sends out roots into the surrounding
medium. (e) One of these roots is transplanted to
another kind of medium, where it forms a shoot.
This plantlet grows into a transgenic tobacco
plant that can be tested for expression of the
transplanted gene.
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Summary Molecular biologists can clone
hundreds of thousands of base pairs of DNA at a
time in yeast artificial chromosomes (YACs). If
they wish to transfer cloned genes to plants,
creating transgenic organisms with altered
characteristics, they use a plant vector such as
the Ti plasmid.