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Transcript
Recombinant DNA
Andy Howard
Introductory Biochemistry
14 October 2010
Biochem: Recombinant DNA
10/14/2010
Recombinant DNA

Much of our current understanding of
molecular biology, and of the ways we
can use it in medicine, agriculture, and
basic biology, is derived from the kinds of
genetic manipulations that we describe
as recombinant DNA
Biochem: Recombinant DNA
10/14/2010
Page 2 of 60
What we’ll discuss






High levels of DNA
structure (concluded)
Synthesis of DNA in
the laboratory
Cloning
Plasmids & inserts
Vector techniques
Libraries & probes
Biochem: Recombinant DNA




High-throughput
Expression
Fusion Proteins
Protein-protein
interactions
10/14/2010
Page 3 of 60
Nucleosome
structure




Core octamer is two molecules
each of H2A, H2B, H3, H4
Typically wraps around
~200bp of DNA
DNA between
nucleosomes is ~54 bp long
H1 binds to linker and to core
particle; but in beads-on-a-string
structure, it’s often absent
Biochem: Recombinant DNA
10/14/2010
Page 4 of 60
How much does this coil up?





200 bp extended would be about 50nm
The width of the core-particle disk is
5nm
So this is a tenfold reduction
Nucleosomal organization corresponds
to negative supercoiling
… so DNA ends up supercoiled when
we take away the histones
Biochem: Recombinant DNA
10/14/2010
Page 5 of 60
Courtesy answers.com
Next level of
organization



H1 interacts with DNA
along linker region
Individual histones
spiral along to form
30 nm fiber
See fig.19.25
Courtesy
Johns
Hopkins
Univ
Biochem: Recombinant DNA
10/14/2010
Page 6 of 60
Even higher…




The 30nm fibers are attached to an
RNA-protein scaffold that holds the
30nm fibers in large loops
Typical chromosome has ~200 loops
Loops are attached to scaffold at their
base
Ends can rotate so it can be
supercoiled
Biochem: Recombinant DNA
10/14/2010
Page 7 of 60
What about prokaryotes?
No actual histones
 Histone-like proteins (HLPs)
involved
 Bacterial DNA attached to scaffold
in large loops (~100kb)
 This makes a nucleoid

Biochem: Recombinant DNA
10/14/2010
Page 8 of 60
How many loops in bacteria?




Typical bacterial genome (E.coli) has
3000 open reading frames ~ 3000 genes.
Assume 500 amino acids per protein =
1500 bases per gene (ignores
transcriptional elements)
Then genome is 1500 bp/gene * 3000
genes = 4.5*106 base-pairs
That’s (4.5*106 bp)/(1*105 bp/loop) = 45
loops
Biochem: Recombinant DNA
10/14/2010
Page 9 of 60
iClicker quiz, question 1

1. How does acetylation of histones
affect their charge state?




(a) It makes them more positively charged
(b) It makes them less positively charged
(c) It does not change their charge state
(d) It depends on whether these are bacterial
histones or eukaryotic histones
Biochem: Recombinant DNA
10/14/2010
Page 10 of 60
iClicker quiz, question 2

2. Suppose a mutation in the gene coding for
histone H1 makes it fold up incorrectly. How will
this mutation influence DNA organization?





(a) It will prevent formation of nucleosomes
(b) It will interfere with the beads-on-a-string
organization between nucleosomes
(c) It will interfere with higher-level organization
involving assembly of solenoids into loops
(d) All of the above
(e) None of the above
Biochem: Recombinant DNA
10/14/2010
Page 11 of 60
Synthesizing nucleic acids




Laboratory synthesis of nucleic acids
requires complex strategies
Functional groups on the monomeric
units are reactive and must be blocked
Correct phosphodiester linkages must
be made
Recovery at each step must high!
Biochem: Recombinant DNA
10/14/2010
Page 12 of 60
Solid Phase
Oligonucleotide Synthesis

Dimethoxytrityl group blocks the 5'-OH
of the first nucleoside while it is linked
to a solid support by the 3'-OH


Step 1: Detritylation by trichloroacetic acid
exposes the 5'-OH
Step 2: In coupling reaction, second base is
added as a nucleoside phosphoramidate
Biochem: Recombinant DNA
10/14/2010
Page 13 of 60
Synthesis I

Figure 11.29
Solid phase oligonucleotide synthesis. The four-step
cycle starts with the first base in nucleoside form (N1) attached by its 3'-OH group to an insoluble, inert
resin or matrix, typically either controlled pore glass
(CPG) or silica beads. Its 5'-OH is blocked with a
dimethoxytrityl (DMTr) group (a).
Biochem: Recombinant DNA
10/14/2010
Page 14 of 60
Blocking
groups
If the base has reactive -NH2
functions, as in A, G, or C,
then N-benzoyl or Nisobutyryl derivatives are
used to prevent their
reaction (b). In step 1, the
DMTr protecting group is
removed by trichloroacetic
acid treatment. Step 2 is
the coupling step: the
second base (N-2) is added
in the form of a nucleoside
phosphoramidite derivative
whose 5'-OH bears a DMTr
blocking group so it cannot
polymerize with itself (c).
Biochem: Recombinant DNA
10/14/2010
Page 15 of 60
Solid Phase Synthesis


Step 3: capping with acetic anhydride
blocks unreacted 5’-OHs of N-1 from further
reaction
Step 4: Phosphite linkage between N-1 and
N-2 is reactive and is oxidized by aqueous
iodine to form the desired, and more stable,
phosphate group
Biochem: Recombinant DNA
10/14/2010
Page 16 of 60
Activation of the
phosphoramidate
Biochem: Recombinant DNA
10/14/2010
Page 17 of 60
Cloning

Cloning is the process whereby DNA is
copied in a controlled way to produce
desired genetic results
Biochem: Recombinant DNA
10/14/2010
Page 18 of 60
Plasmids



Small (typically < 10 kbp), usually
circular segments of DNA that get
replicated along with the organism’s
chromosome(s)
Bacterial plasmids have a defined
origin of replication and segments
defining specific genes
Some are natural;
others are man-made
Biochem: Recombinant DNA
10/14/2010
Page 19 of 60
How they’re used


Typical man-made plasmid includes a gene
that codes for an enzyme that renders the
bacterium resistant to a specific antibiotic,
along with whatever other genetic materials
the experimenter or clinician wishes to
incorporate
Thus the cells that have replicated the
plasmid will be antibiotic-resistant; surviving
colonies will be guaranteed (?) to contain the
desired plasmid in all its glory
Biochem: Recombinant DNA
10/14/2010
Page 20 of 60
A typical
plasmid
Biochem: Recombinant DNA
10/14/2010
Page 21 of 60
Building useful
plasmids


Take starting plasmid and
cleave it with a restriction
enzyme at a specific site
Add foreign DNA that has
been tailored to fit into that
plasmid
Biochem: Recombinant DNA
10/14/2010
Page 22 of 60
Inserts

Typically a place within the plasmid will
be set up so that small stretches (< 10
kbp) of desired DNA can be ligated in


With sticky ends: high specificity, but you do
get self-annealing of the plasmid and of the
insert, so those have to be eliminated
With blunt ends: require more artisanry:
T4 phage ligase can rejoin ends without
stickiness; but it’s chaotic
Biochem: Recombinant DNA
10/14/2010
Page 23 of 60
Directional
cloning

Guarantees
that the
desired DNA
goes in in
exactly one
orientation
Biochem: Recombinant DNA
10/14/2010
Page 24 of 60
Use of
bacteriophage
lambda


Can handle somewhat
larger inserts (10-16
kbp)
Middle third of its
48.5-kbp chromosome
isn’t needed for
infection
Biochem: Recombinant DNA
10/14/2010
Page 25 of 60
Cosmids

14-bp sequence cos (cohesive end site):
5’-TACGGGGCGGCGACCTCGCG-3’
3’-ATGCCCCGCCGCTGGAGCGC-5’

… one of these at each end
Must be 36 kbp < separation < 51 kbp
apart
In practice we can use these for inserts
up to 40 kbp in size


Biochem: Recombinant DNA
10/14/2010
Page 26 of 60
Cosmids in
action
(fig. 12.9)
Biochem: Recombinant DNA
10/14/2010
Page 27 of 60
Shuttle vectors




These are plasmids that can operate
in two different organisms
Usually one prokaryote and one
eukaryote (e.g. Escherichia coli and
Saccharomyces cerevisiae)
Separate origins for each host
This allows us to clone the vector in a
bacterial host and then express it in a
eukaryotic setting
Biochem: Recombinant DNA
10/14/2010
Page 28 of 60
Typical shuttle vector
Biochem: Recombinant DNA
10/14/2010
Page 29 of 60
Artificial chromosomes



Huge chunks (2 megaBp!) can be
propagated in yeast with artificial
chromosomes (YACs)
These can be manipulated in the yeast
setting or transferred to transgenic mice
in a living animal
YACs need origin, a centromere, and
telomeres
Biochem: Recombinant DNA
10/14/2010
Page 30 of 60
Use of YACs in mice

QuickTime™ and a
decompressor
are needed to see this picture.
Biochem: Recombinant DNA
10/14/2010
Diagram
courtesy
Expert
Reviews
in
Molecular
Medicine,
2003
Page 31 of 60
DNA libraries




Set of cloned fragments that make up an
organism’s DNA
We can isolate genes from these
Most common approach to creating these is
shotgun cloning, in which we digest the total
DNA and then clone fragments into vectors
Goal is that >= 1 clone will contain at least part
of the gene of interest (might have been clipped
by the restriction enzyme!)
Biochem: Recombinant DNA
10/14/2010
Page 32 of 60
Probabilities




Probability P that some number of
clones, N, contains a particular
fragment representing a fraction f of
the genome:
P = 1 - (1 - f)N
Therefore 1-P = (1-f)N
Thus ln(1-P) = ln{(1-f)N} = Nln(1-f)
Therefore N = ln(1-P) / ln(1-f)
Biochem: Recombinant DNA
10/14/2010
Page 33 of 60
What that means



The value f is pretty small, so the
denominator is only slightly negative;
whereas we want the numerator to be
very negative, since that corresponds
to a high value of P.
10 kbp fragments in E.coli means
f = 10/4640 = 0.0022,
so for P = 0.99, we need N=1.4*106
We’d do better with larger f values!
Biochem: Recombinant DNA
10/14/2010
Page 34 of 60
Finding relevant fragments
by colony hybridization





Plate out a library of fragments and grow
colonies or plaques
Soak those onto a flexible absorbent disc
Disc is treated with high-pH to dissociate bound
DNA duplexes; placed in a sealed bag with a
radiolabeled probe
If they hybridize, radioactivity will stick to disc
The hits can be recovered from the master plate
Biochem: Recombinant DNA
10/14/2010
Page 35 of 60
Colony
hybridization
illustrated
Biochem: Recombinant DNA
10/14/2010
Page 36 of 60
Making the probes





Sometimes we have at least part of the gene
sequence and can fish for it
Other times we know the amino acid sequence
and can work backward, but with degeneracy
(64 codons, 20 aa’s)
Typically use at least 17mers to guarantee that
the don’t get random association
Probes derived from a different species are
heterologous
With big eukaryotic genes we may have to look
for pieces of the gene, not the whole thing
Biochem: Recombinant DNA
10/14/2010
Page 37 of 60
cDNA libraries



Sometimes the easiest thing to obtain
are mRNA templates associated with a
particular function
Reverse transcriptase can make a
complementary (cDNA) molecule from
such an mRNA template
A library of cDNAs can be assembled
from a collection of mRNA templates
Biochem: Recombinant DNA
10/14/2010
Page 38 of 60
Why is that useful?



The mRNAs will be unique to the cell
type from which they’re derived
Often they’re also unique to the
functional role that tissue is playing
at the time
Therefore finding that collection of
DNA tells us about cellular activity
Biochem: Recombinant DNA
10/14/2010
Page 39 of 60
Expressed sequence tags



An EST is a short (~200 base) sequence
derived from a cDNA
Represents part of a gene that is being
expressed
Labeled ESTs can be mounted on a gene
chip and used to identify cells that are
expressing a particular class of mRNAs
Biochem: Recombinant DNA
10/14/2010
Page 40 of 60





Southern blots I:
fractionation
Tool for identifying a particular DNA
fragment out of a vast population thereof
Exploits sequence specificity for
identification
Developed by E.M.Southern in 1975
Begins with electrophoretic fractionation
of fragments (mobility  1/mass)
Polyacrylamide gels ok 25-2000 bp;
agarose better for larger fragments
Biochem: Recombinant DNA
10/14/2010
Page 41 of 60
Southern blots 2: blotting






Gel soaked in base to denature duplexes
pH readjusted to neutral
Sheet of absorbent material placed atop the gel
Salt solution is drawn across the gel, perp to the
electrophoretic direction, in various ways to carry
the DNA onto the sheet
Sheet is dried in an oven to tightly attach the DNA
to it
Incubate sheet with protein or detergent to
saturate remaining DNA binding sites on sheet so
we don’t get nonspecific binding
Biochem: Recombinant DNA
10/14/2010
Page 42 of 60
Southern blots 3:
hybridization



Labeled probe and sheet placed in
sealed bag
If probe attaches, label will appear at
that point on the sheet via annealing
or hybridization
Label detected by autoradiography
Biochem: Recombinant DNA
10/14/2010
Page 43 of 60
Southern
blots
illustrated
Biochem: Recombinant DNA
10/14/2010
Page 44 of 60
Variations on this idea




RNA can be used as the probe: that’s
called a Northern blot
Proteins can be substituted by using
an antibody as a probe and a
collection of protein fragments as the
analytes; that’s called a Western blot
Ha ha
There is no Eastern blot, as far as I
know.
Biochem: Recombinant DNA
10/14/2010
Page 45 of 60
High-throughput
techniques


Eagerness to provide rapid, easy-to-use
applications of these approaches has led
to considerable research on ways to
make these techniques work fast and
automatically
This high-throughput approach enables
many experiments per unit time or per
dollar
Biochem: Recombinant DNA
10/14/2010
Page 46 of 60
DNA microarrays




Thousands of oligonucleotides
immobilized on a substrate
Synthesis by solid-phase
phosphoramidite chemistry
Typically 25-base oligos
Can be used in cDNA projects to look at
expression patterns
Biochem: Recombinant DNA
10/14/2010
Page 47 of 60
An
example
Biochem: Recombinant DNA
10/14/2010
Page 48 of 60
Using expression vectors



We often want to do something with
cloned inserts in expression vectors, viz.
make RNA or even protein from it
RNA: stick an efficient promoter next to
the cloning site; vector DNA transcribed
in vitro using SP6 RNA polymerase
This can be used as a way of making
radiolabeled RNA
Biochem: Recombinant DNA
10/14/2010
Page 49 of 60
Protein expression




Making (eukaryotic) proteins in bacteria via
cDNA means we don’t have to worry about
introns
Expression vector must have signals for
transcription and translation
Sequence must start with AUG and include a
ribosome binding site
Strong promoters can coax the bug into
expressing 30% of E.coli’s protein output to be
the one protein we want!
Biochem: Recombinant DNA
10/14/2010
Page 50 of 60
QuickTime™ and a
decompressor
are needed to see this picture.
Example: ptac


This is a fusion of lac promoter (lactose
metabolism) with trp promoter
(tryptophan biosynthesis)
Promoter doesn’t get turned on until an
inducer (isopropyl--thiogalactoside,
IPTG) is introduced
Biochem: Recombinant DNA
10/14/2010
Page 51 of 60
iClicker quiz, question 3






Probe systems employing RNA are called
(a) Southern blots
(b) Northern blots
(c) Western blots
(d) Eastern blots
(e) None of the above
Biochem: Recombinant DNA
10/14/2010
Page 52 of 60
iClicker quiz, question 4

4. The inducer used with the ptac
promoter system is





(a) glucose
(b) glucose-6-phosphate
(c) IPTG
(d) ionizing radiation
(e) none of the above.
Biochem: Recombinant DNA
10/14/2010
Page 53 of 60
Eukaryotic expression




Sometimes we need the glycosylations and
other PTMs that eukaryotic expression enables
This is considerably more complex
Common approach is to use vectors derived
from viruses and having the vector infect cells
derived from the virus’s host
Example: baculovirus, infecting lepidopteran
cells; gene cloned just beyond promoter for
polyhedrin, which makes the viral capsid protein
Biochem: Recombinant DNA
10/14/2010
Page 54 of 60
Screening libraries
with antibodies





Often we have antibodies that react with a protein of
interest
If we set up a DNA library and introduce it into host
bacteria as in colony hybridization, we can put nylon
membranes on the plates to get replicas of the
colonies
Replicas are incubated to make protein
Cells are treated to release the protein so it binds to
the nylon membrane
If the antibody sticks to the nylon, we have a hit!
Biochem: Recombinant DNA
10/14/2010
Page 55 of 60
Fusion proteins




Sometimes it helps to co-express our protein of
interest with something that helps expression,
secretion, or behavior
We thereby make chimeric proteins, carrying
both functionalities
We have to be careful to keep the genes in
phase with one another!
Often the linker includes a sequence that is
readily cleaved by a commercial protease
Biochem: Recombinant DNA
10/14/2010
Page 56 of 60
Fusion systems (table 12.2+)
Product
Origin
Mass, Secre Affinity Ligand
kDa
ted?
-galactosidase
E.coli
116
No
APTG
Protein A
Staph.
31
Yes
IgG
Chloramphenicol
acetyltransferase
E.coli
24
Yes
Chloramphenicol
Streptavidin
Strep.
13
Yes
Biotin
Glut-S-transferase E.coli
26
No
Glutathione
Maltose Bind.Prot. E.coli
40
Yes
Starch
Hemoglobin
16/32
No
None
Vitreoscilla
Biochem: Recombinant DNA
10/14/2010
Page 57 of 60
Improving purification
via expression




If we attach (usually at the N-terminal end) a his-tag
(several his, several cys) to our protein, it becomes
easier to purify:
The his tag forms a loop that will bind strongly to a
divalent cation like Ni2+
Thus we can pour our expressed protein through a
Ni2+ affinity column and it will stick, while other
proteins pass through
We elute it off by pouring through imidazole, which
completes for the Ni2+ and lets our protein fall off
Biochem: Recombinant DNA
10/14/2010
Page 58 of 60
Protein-protein interactions


One of the key changes in biochemistry over
the last two decades is augmentation of the
traditional reductionist approach with a more
emergent approach, where interactions
among components take precedence over
the properties of individual components
Protein-protein interaction studies are the
key example of this less determinedly
reductionist approach
Biochem: Recombinant DNA
10/14/2010
Page 59 of 60
Two-hybrid screens



Use one protein as bait; screen many
candidate proteins to see which one
produces a productive interaction with
that one
Thousands of partnering relationships
have been discovered this way
Some of the results are clearly
biologically relevant; others less so
Biochem: Recombinant DNA
10/14/2010
Page 60 of 60