Transcript Cloning Overview

Cloning
Overview
• Foreign DNA is prepared for
insertion into a vector DNA
– Both foreign and vector DNA are
cut with a restriction enzyme
– The restriction enzyme leaves
sticky ends
• Short regions of homology at the
site of cleavage
– The foreign DNA and vector DNA
are mixed together
• The sticky ends bind DNA
together
– DNA ligase reseals the double
helices
• DNA is taken up by host cells
• As the host cells are grown, the
recombinant DNA grows with
them
Libraries are made
when DNA is cloned
indiscriminately
– All possible DNA from
a foreign source is
inserted into vectors
one fragment at a time
– Bacteria take up the
vectors en masse one
vector per bacterium
– Propagation of the
bacteria creates a
culture that contains all
of the DNA from a
foreign source,
fragmented into small
pieces, with each piece
in its own bacterial
clone
Libraries
Three general types of libraries
• Conceptually represent DNA, RNA and protein libraries
– Genomic libraries
• This is used to clone genes
– cDNA libraries
• Made from mRNA, this contains sequence representing message
– Expression libraries
• A cDNA library that expresses the foreign proteins
Genomic Library
• A genomic library contains all of the
chromosomal DNA of a cell
• DNA is purified and fragmented into pieces
ranging from a few thousand bases to hundreds of
thousands
– The size of the fragments depends on the capacity of
the vector to contain and propagate the DNA
– The fragments are ligated into a vector and the vector
propagated in a suitable host cell culture
– Each piece of chromosomal DNA is then grown within
a foreign host cell
cDNA Libraries
• A cDNA library contains the sequences of
(primarily) mRNA found in a cell
– These sequences are propagated following conversion of
a single stranded RNA molecule into double stranded
DNA through the action of reverse transcriptase
– They lack the transcriptional control and intronic
sequences found in genomic clones
– They are useful
• In understanding structure and function of an mRNA
– For example, the nucleotide sequence of an mRNA defines the exons of
a gene
• In expressing eukaryotic proteins in bacteria
Expression libraries
• These are cDNA libraries in a special form of vector
that permits transcription of the incorporated cDNA
– Proteins or protein fragments then appear with bacterial
host cells that are normally not present
– These can be identified based on their antigenicity or
activities
– By this route a cDNA sequence can be isolated based on
identification of its protein product
– Proteins can also be made in quantity and purified more
easily when made in bacteria
Why
clone?
• Analytical purposes
– The DNA sequence and the
structure/function relationships of
a gene can be determined in
isolation from the surrounding
DNA of the genome by purifying
the gene through cloning
• A single gene is lost in the
background of the genome
• Cloning it isolates it.
• Practical purposes
– An isolated gene can be
• Expressed to produce a protein in
vivo or in vitro
– Commercial purposes
– Therapeutic purposes
• Manipulated to change its
sequence
– This makes new genes with
original proteins and properties
– Cloning creates an unlimited
supply of identical copies
Cloning reagents
• Enzymes and buffers
– Restriction enzymes
– DNA ligase
– And sometimes (for cDNA clones)
• Reverse transcriptase and its allied reagents
• Vectors
• Host cells
• And
– Microbiological supplies
– Radioactive or fluorescence labeled nucleotides
DNA
ligase
• While we have looked at ligase
as an enzyme used during DNA
replication, DNA ligase used
during cloning procedures
comes from the bacterial virus
T4
• The enzyme is fragile and
reactions are carried out at low
temperature
• It is capable of resealing the cut
ends of restricted DNA,
including blunt ends
• Typically DNA is not purified
away from restriction enzymes,
but simply heated following
restriction to denature the
restriction enzyme
– Then ligation is not defeated by
recutting
Reverse
transcriptase
• This is used for converting an
RNA transcript into DNA such
that it can be cloned.
– This is called copy DNA or cDNA
• mRNA is primed with oligo dT
for reverse transcription
– RT makes a single stranded DNA
copy of the RNA
• The RNA is completely
degraded and several strategies
are employed to prime second
strand synthesis
– A hairpin loop naturally forms
• But this loses sequence on the 5’
end
– Oligo dA or dG can be
enzymatically polymerized from
the 3’ end
• Then oligo dT or dC can be used
as with the first strand
• The hairpin loop and ragged
ends of the new duplex cDNA
are digested with a single strand
specific nuclease making the
duplex blunt ended
• Then “linker” DNA is ligated
onto the duplex
– Linker DNA is a palindromic
duplex oligonucleotide with a
blunt end recognized by a
restriction enzyme
• Here it is HindIII
– The linker DNA is then digested
(with Hind III in this example) and
the resulting DNA represents
• The mRNA
• A Hind III sticky end
– This can be ligated into a vector as
though it were genomic DNA that
had simply been fragmented by
Hind III
Vectors
• DNA with an independent origin of replication and
some selectable or differential markers
– A selectable marker permits a host cell to survive in
otherwise lethal environments
– A differential, non-selectable marker permits
identification of a bacterium by its appearance
– Plasmids, viruses (and viral derivatives) and artificial
chromosomes
• All forms of cloning are technical variations on plasmid cloning
PBR322
• This is an artificial plasmid
vector that has educational
value, but is rarely used
anymore
– Commercial plasmid vectors
are more versatile
• PBR322 has two genes for
antibiotic resistance
– Amp and tet
• There is a single site for the
restriction enzyme Bam H1
in the tet gene
– Inserting foreign DNA into
this site inactivates the gene
The host
• May be prokaryotic or
eukaryotic
• Must take up recombined DNA
– Technical approaches to
introducing DNA into bacterial
cells
• Add DNA to cells directly
– Transformation
» Bacteria take up plasmids
– Transfection
» Bacteria take up viral
vectors
» Eukaryotes take up any
vectors
• Electroporation
– Drive DNA into cells with
electric field
• Must support growth of the
recombined DNA
Colony
screens
• Once recombinant DNA has
been taken up by a host, a
successful transformation
needs to be identified
• Bacterial colonies are grown
on a nutrient plate
– If the foreign DNA was purified
before it was inserted into the
vector, then the selectable
markers provide enough
information to identify
successful clones
– However if the foreign DNA
was not purified, then the
bacterial colonies may
overlayed with a membrane and
lysed in situ on the membrane
• The bacteria and DNA will
stick to the membrane
• Successful clones hybridize to
radioactive complementary
DNA like a southern blot
What to do with the DNA?
• Once recombinant DNA is in a host, the host can
be grown and plasmid easily isolated
• Sequence it
– This can be done directly on purified recombinant DNA
• Express it
– Proteins may be made in quantity
• Mutate it
– Site specific mutations are possible
• Once a sequence is known, it is possible to alter any nucleotide
by design
• New proteins may be designed
• Control elements of the inserted DNA may be studied and
altered
Examples of medical relevance
• Insulin Dependent Diabetes Mellitus (diabetes
type I)
– This results from an autoimmune destruction of the
pancreatic beta cells
• The body can no longer make insulin
– Therapy requires monitoring of blood sugar and
administration of insulin depending on glucose
levels
• Insulin originally came from animal sources
– This molecule eventually elicited an immune response
• Cloning technology made it theoretically possible to
clone human insulin
– There would be no immune response to this
Cloning insulin
• The amino acid sequence of insulin was known, so a synthetic DNA
sequence (probe) complementary to the insulin gene was available
– The gene sequence is not exactly predictable due to the degeneracy of the code,
but close enough to insure a unique identification
– The conditions at which probe is washed off of a membrane are made less harsh
(lowering the stringency of the wash)
• This allowed imperfect hybrids of a sufficiently long probe to survive the wash
cDNA clones
were necessary
• The insulin gene has two introns,
one of which interrupts the coding
sequence of the gene
• In order to express the protein, a
cDNA clone was needed that
eliminated the intron
– Bacteria could not process mRNA
from a cloned eukaryotic gene
• mRNA from an insulinoma
(pancreatic beta cell tumor) was
isolated and cDNA made
– This represented every message in the
cell
• Insulin mRNA represented a fraction
of the total
– All of the cDNA was inserted into
plasmids at once
• It wasn’t possible to purify insulin
mRNA first
• Each vector got a cDNA from a
random mRNA from the insulinoma
Identifying the insulin cDNA
clone
• The culture of bacteria
transformed with the
insulinoma cDNA’s is called a
library
– It represents all of the mRNA in
the cell
• Bacteria were plated and the
colonies “lifted” onto a
membrane
– Probing the membrane with a
synthetic complementary
sequence identified colonies that
contained the insulin cDNA
Expressing the clone
• Once the cDNA was isolated,
the “insert” was removed from
the vector and cloned into a
vector that contained control
sequences for RNA polymerase
– The control sequences were the
lac promoter
– Transcription of the insulin gene
could be increased with IPTG
• Bacteria were transformed with
this recombinant vector and
insulin protein was synthesized
from the transcript polymerized
by RNA polymerase
Problems with expressing
eukaryotic genes in a bacterium
• But the insulin could not be properly
processed in bacteria
– The signal peptide and the center peptide could
not be removed by the bacterium
– It was also difficult to purify because the signal
peptide caused aggregation of the protein
within the bacterium
Another
approach
• The A and B chains represented individual
polypeptides that are normally produced by
processing of preproinsulin
– It was necessary to remove the signal peptide from
the clone prior to expression
• The gene was cloned as a fusion protein with
beta galactosidase
– The gene, lacking the signal sequence but
containing an N terminal methionine was used to
replace the 3’ end of the betagalactosidase gene
– This meant that expression of the cloned fusion
gene produced a protein that was
betagalactosidase on its amino terminal end and
proinsulin (with an extra methionine) on the
carboxyterminal end
• This helped in purification because beta
galactosidase did not aggregate and was easily
purified
• But the insulin part had to be separated from beta
galactosidase
Reconstitution of insulin from
individual chains
• The extra methionine is specifically recognized
and cleaved by the chemical reagent cyanogen
bromide
– This splits the insulin away from the beta galactosidase
• Proinsulin was then mixed with C peptidases, that
are made in pancreatic beta cells
– This freed the A and B chains (now linked through
disulfide bridges) producing insulin
Present day
• The complete cDNA
(including signal peptide)
have been cloned into yeast
• The yeast contain ER and
signal peptidase, and they
have also been engineered
to contain the protease that
cleaves the internal cpeptide form insulin
– The yeast secrete human
insulin
– This circumvents the costly
procedures necessary to
purify insulin away from
bacteria and then from the
beta-gal.
• In immunodeficient mice, IDDM I
can be cured with pancreatic beta
cell transplantation
– But beta cells are killed by an
autoimmune response in people
– Transplanted beta cells would simply be
killed by the immune system
• The use of insulin as a drug could be
circumvented by putting an active
insulin gene inside a patient
• What kinds of problems might you
expect in attempting this?
Gene therapy for
IDDM type I?
– How would you control the gene?
– In what cell type would you put it?
– What other systems in the host cell
would be required for proper expression
of insulin?
• The pancreatic beta cell responds to
elevated blood glucose by releasing stored
insulin through exocytosis
• Properly regulated insulin requires
– Targeting to exocytic vesicles
Beta cell
regulation
• This is due to recognition of preproinsulin
structure within the ER and Golgi
– Recognition systems must be present
– The signal peptide and C-peptide must be
removed
– Storage prior to release
• Exocytic vesicles must form containing mature
insulin
– They must be sequestered until a signal for
release is received
– Responsiveness of exocytosis to blood glucose
• Exocytosis involves elevated calcium levels that
promote fusion of the exocytic vesicle with the
plasma membrane
Other human diseases potentially
amenable to gene therapy
• Most active
– Severe combined immune deficiency (SCID)
• Especially deficiency of adenosine deaminase
• The expression of the gene needn’t be controlled and is expressed in rapidly
growing cells (stem cells of the hematopoietic system)
• Can’t find or transfect stem cells?
– Cystic fibrosis
•
•
•
•
Lack of a chloride channel
Also expression needn’t be controlled
Accessible target cells (alveolar cells create the main clinical problem)
Delivery systems inadequate or unstable DNA?
• Probability for a therapy to work increases if the expression
levels of the protein don’t matter and that the defect is due
to a missing enzyme