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• 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

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 c-peptide 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.

Gene therapy for IDDM type I?

• 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?– 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?

Beta cell regulation

• The pancreatic beta cell responds to elevated blood glucose by releasing stored insulin through exocytosis

• Properly regulated insulin requires– Targeting to exocytic vesicles

• 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