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7 Cloning Vectors forEukaryotes

7 Cloning Vectors forEukaryotes

Vectors for yeast and other fungiCloning vectors for higher plantsVectors based on animal viruses

Cloning vectors for organismsother than E. coli

Cloning vectors for organismsother than E. coli

• Most cloning experiments are carried outwith E. coli• Study basic features of molecular biology

• Biotechnology• Use cloning to control or improve synthesis of

an important metabolic product• Change properties of the organism (E. coli)

• Therefore consider

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7.1 Vectors for yeast and otherfungi

7.1 Vectors for yeast and otherfungi

• Saccharomyces cerevisiae• one of most important organisms in

biotechnology• 2 mm plasmid:

• one of only a very limited number of plasmidsfound in eukaryotic cells

2 mm plasmid2 mm plasmid

• An excellent basis for a cloning vector• 6 kb in size, copy number of 70-200• Replication makes use of a plasmid origin

• All is not perfectly straightforward in using the 2mm plasmid.• => There is the question of a selectable marker

Figure 7.1 REP1 and REP2 are involvedin replication of the plasmid, and FLPcodes for a protein that can convert the Aform of the plasmid to the B form, inwhich the gene order has been rearrangedby intramolecular recombination. Thefunction of D is not exactly known.

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7.1.1. Selectable markers for the 2mm plasmid

7.1.1. Selectable markers for the 2mm plasmid

• Gene LEU2• b -isopropyl malate dehydrogenase

• An auxotrophic mutant• Special host that has a non-functional LEU2 gene• Such a leu2- yeast is unable to synthesize leucine• survive only if this amino acid is supplied

Using LEU2 as a selectablemarker

Using LEU2 as a selectablemarker

• Cells are plated out onto minimal medium• Which contains no added amino acids

• Only transformed cells are able to survive andform colonies

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7.1.2. Vectors based on the 2 mm circle -yeast episomal plasmids (YEps)7.1.2. Vectors based on the 2 mm circle -yeast episomal plasmids (YEps)

• Yeast episomal plasmids or YEps• Vectors derived from 2 mm plasmid• Some YEps contain entire 2 mm plasmid, others

include just 2 mm origin of replication

A yeast episomal plasmid -YEp13

A yeast episomal plasmid -YEp13

• It is a shuttle vector• 2 mm origin of replication• Selectable LEU2 gene• Entire pBR322 sequence• Can replicate & be selected

for in both yeast & E. coli

• One of reasons• Difficult to recover

recombinant DNA moleculefrom a transformed yeastcolony

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The standardprocedure of cloning

with YEp13

The standardprocedure of cloning

with YEp13• Perform initial

cloning experimentwith E. coli

• Selectrecombinants inyeast

7.1.3. A YEp may insert into yeastchromosomal DNA

7.1.3. A YEp may insert into yeastchromosomal DNA

• Episomal• A Yep can replicate as

an independent plasmid• carry an origin of

replication• Integration into one of

yeast chromosomes canoccur

• Insertion of entire plasmid intoyeast chromosomes

v Recombination can occurbetween plasmid LEU2gene & yeast mutant LEU2gene

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7.1.4. Other types of yeastcloning vector

7.1.4. Other types of yeastcloning vector

Yeast integrative plasmids (YIps)Yeast replicative plasmids (YRps)

Yeast integrative plasmids (YIps)Yeast integrative plasmids (YIps)

• Selectable marker: URA3• orotidine-5'-phosphate

decarboxylase• Biosynthesis pathway for

pyrimidine nucleotides

• YIp5• pBR322+ URA3A

gene

n YIp cannot replicate as a plasmidas it does not contain any parts of 2mm plasmid

n Instead depends for its survival onintegration into yeast chromosalDNA

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Yeast replicative plasmids (YRps)Yeast replicative plasmids (YRps)

• Are able to multiply asindependent plasmids

• They carry an origin ofreplication

• Sele ctable ma rker:TRP1

• Is involved intryptophan biosynthesis

• Is located adjacent to achromosomal origin ofreplication

• YRp7• pBR322+TRP1

7.1.4. Other types of yeast cloningvector

7.1.4. Other types of yeast cloningvector

• Transformation frequency• Number of transformants that can be obtained per mg of

plasmid DNA• YEps: 10,000-100,000• YRps: 1000-10,000• YIp: <1000, and only 1-10 unless special procedures

are used• Copy number

• YEps & YRps: 20-50 and 5-100 respectively• Unstable recombinants

• YIp: one copy per cell• Stable recombinants

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7.1.5. Artificial chromosomescan be used to clone large

pieces of DNA in yeast

7.1.5. Artificial chromosomescan be used to clone large

pieces of DNA in yeast

Structure of eukaryoticchromosomes

Structure of eukaryoticchromosomes

• The centromere• Chromosomes to be

distributed correctly• Two telomeres

• Ends to be replicatedcorrectly

• Prevent chromosome fromexonucleases

• The origins of replication• DNA replication initiates

Figure 7.7 Chromosomestructure

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The structure and use of a YACvector

The structure and use of a YACvector

• Selectable markers:• TRP1, URA3 & SUP4

• TRP1-origin-CEN4fragment: centromere

• TEL: telomeres

(a) pYAC3: a pBR322

• Yeast auxotrophic mutant:

• YAC only:

Cloning strategy with pYAC3Cloning strategy with pYAC3

SnaBI: a blunt cutter, TACGTA

SUP4+

Red colonies

Recombinants:

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Application for YAC vectorsApplication for YAC vectors

• YACs opened way to studies functions &modes of expression of genes• > 150 kb, human cystic fibrosis gene = 250 kb• YACs can be propagated in mammalian cells

• YACs are equally important in theproduction of gene libraries• E. coli vector: 300 kb x 30,000 clones• YAC vector: 600-1400 kb x 6500 clones

• Large-scale DNA sequence in HumanGenome Project

7.1.6 Vectors for other yeastsand fungi

7.1.6 Vectors for other yeastsand fungi

• 2 mm plasmid are able to replicate in a fewother types of yeast

• The range of species is not broad enough for 2 mmvectors to be general value

• Requirements of biotechnology are betterserved by integrative plasmids: stablerecombinants

• Efficient integrative vectors are now available for anumber of species

• Yeast such as Pichiapastoris and Kluveromyces lactis,& filamentous fungi Aspergillus nidulans & Neurospracrassa

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7.2 Cloning vectors for higherplants

7.2 Cloning vectors for higherplants

• Cloning vectors for higher plants weredeveloped in the 1980s• Genetically modified (GM) crops• That are in headlines today

• Three types of vectors system• Vectors based on naturally occurring plasmids

of Agrobacterium.• Direct gene transfer using DNA fragments not

attached to a plant cloning vector• Vectors based on plant viruses

Agrobacterium tumefaciensAgrobacterium tumefaciens

nature’s smallest genetic engineer

A soil microorganismCause crown gall disease

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Agrobacteriumtumefaciens

Agrobacteriumtumefaciens

After infectionn A cancerous

proliferation ofstem tissue

Figure 7.10 The Ti plasmid & its intergrationinto the plant chromosomal DNA after A.

tumefacients infection

Figure 7.10 The Ti plasmid & its intergrationinto the plant chromosomal DNA after A.

tumefacients infection

(a) A Ti plasmid• Ability to cause crown gall disease is associated with

the presence of Ti (tumor inducing) plasmid withinbacterial cell

A large plasmid

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Figure 7.10 (b) Integration of T-DNAinto plant genome

Figure 7.10 (b) Integration of T-DNAinto plant genome

• After infection• Part of Ti plasmid is integrated into plant chromosome• T-DNA: 15-30 kb in size, eight or so genes

Figure 7.10 (c) Expression of T-DNAgene

Figure 7.10 (c) Expression of T-DNAgene

• Responsible for cancerous properties oftransformed cells

• Direct synthesis of opines: nutrients of bacteria

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Using the Ti plasmid to introducenew genes into a plant cell

Using the Ti plasmid to introducenew genes into a plant cell

• The large size of Ti plasmid makes itsmanipulation very difficult• A unique restriction site is an imposibility

1) The binary vector strategy2) The cointegration strategy

The binary vector strategyThe binary vector strategy

Figure 7.11 Plasmids A & B complement each other whenpresent together in the same A. tumefaciens cell. The T-DNA carried by plasmid B is transferred to the plantchromosome DNA by proteins coded by genes carried byplasmid A.

• The T-DNAplasmid (B) issmall enough tohave a uniquerestriction site

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Thecointegration

strategy

Thecointegration

strategy

• A new plasmidcarrying a smallportion of T-DNA

• Recombination

Production of transformedplants with the Ti plasmidProduction of transformedplants with the Ti plasmid

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(a) Wound infection byrecombinant A. tumefaciens

(a) Wound infection byrecombinant A. tumefaciens

• By infection of a wound in stem• Only cells in resulting crown gall will possess

cloned gene• Little value to biotechnologist

(b) Transformationof cultured plant

cells

(b) Transformationof cultured plant

cells

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Strategies for introducing newDNA into plant cells

Strategies for introducing newDNA into plant cells

• Figure 5.14 (b) Transformation of plantprotoplasts

• Protoplasts:• Enzyme that degrade yeast, fungal & plant cell walls

are available

The “disarmed” Ti vectors:transformed cells do not display cancerous properties

The “disarmed” Ti vectors:transformed cells do not display cancerous properties

• All cancer genes lie in T-DNA & are notneeded for infection process

• T-DNA involved in infection• Two 25 bp repeat sequence at left & right

borders of region integrated into plant DNA• Disarmed: remove all cancer genes

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Figure 7.14 The binary Ti vectorpBIN19

Figure 7.14 The binary Ti vectorpBIN19

• A number of disarmedTi vectors are nowavailable• A shuttle vector• Initial manipulations

are carried out in E.coli

• Then, correctrecombinant pBIN19being transferred to A.tumefaciens & thenceinto plant

• kanR=kanmycin resistancegene

The Ri plasmidThe Ri plasmid

• The Ri plasmid of Agrobacteriumrhizogenes

• Main difference between Ri & Ti plasmids• Transfer of T-DNA from an Ri plasmid to a

plant results in hairy root disease• Massive proliferation of a highly breached root

system• Growing transformed roots at highly density

in liquid culture• Obtaining large amounts of protein

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Limitations of cloning withAgrobacterium plasmids

Limitations of cloning withAgrobacterium plasmids• In nature A. tumefaciens & A. rhizogenes infect

only dicotyledonous plants• Monocots are outside of normal host range• OK: tomato, tobacco, potato, peas & beans• No: wheat, barley, rice & maize

• Bombardment with microprojectiles• Introduce plasmid DNA directly into plant embryos• Successful with maize & several other important

monocots• Figure 5.15 (b)

7.2.2 Cloning genesin plants by direct

gene transfer

7.2.2 Cloning genesin plants by direct

gene transfer

• A supercoiled plasmid• Unable to replicate in

a plant cell on its own• Become integrated by

recombination intoone of plantchromosomes

• The recombinationevent is poorlyunderstood

Integration appearsto occur randomlyat any position inany of plantchromosome

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(a) precipitation of DNA ontosurfaces of protoplasts

(a) precipitation of DNA ontosurfaces of protoplasts

• Resuspending protoplasts in a viscous solution ofpolyethylene glycol• a polymeric, negatively charged compound• precipitate DNA onto surfaces of protoplasts• induce uptake by endocytosis

(b) fusion of protoplasts with DNA-containing liposomes

(b) fusion of protoplasts with DNA-containing liposomes

• Protoplast can also befused with DNA-containing liposomes

• Intact cells can bevigorously shakenwith DNA-coatedsilica needles• Which penetrate the

cell wall and transferthe DNA into theinterior

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Attempts to use plant virussesas cloning vectors

Attempts to use plant virussesas cloning vectors

• One Problem• Vast majority of plant viruses have genome not of DNA

but of RNA• RNA viruses are nor so useful as potential cloning

vectors• Manipulations with RNA are rather more difficult to

carry out• Only two classes of DNA virus infect higher

plants• Caulimovirus vector & Geminivirus vectors• Neither is ideally suited for gene cloning

7.3 Cloning vectors for animalcells

7.3 Cloning vectors for animalcells

• Synthesis of recombinant protein fromgene• That are not expressed correctly in E. coli or

yeast• Devise techniques for gene therapy

• A disease is treated by introduction of a clonedgene into the patient

• Cloning in insects is interesting• It makes use of a novel type of vector that we

have not met so far

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7.3.1 Cloning vectors for insects7.3.1 Cloning vectors for insects

• Fruit fly, Drosophila melanogaster• One of the most important model organisms

used by biologists• More recently, homeotic selector genes

• Genes control overall body plan of fruitfly• Closely related to equivalent genes in

mammals• Led to D. melanogaster being used as a model

for the study of human developmentalprocesses

P elements as cloning vectorsfor Drosophila

P elements as cloning vectorsfor Drosophila

• No plasmids are know in Drosophila• P element in Drosophila

• 2.9 kb transposon• 3 genes flanked by 2 short inverted repeat sequences• Genes: transposase, enzyme carries out the

transposition process• Inverted repeats form the recognition sequences

(a) The structure of a P element

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P elements as cloning vectorsfor Drosophila

P elements as cloning vectorsfor Drosophila

• P elements• Move from one site to another within a single

chromosome• Jump between chromosomes• Between a plasmid & one of fly’s chromosomes

(b) P element transposition

(c) The structure of a P elementcloning vector

(c) The structure of a P elementcloning vector

• One P elements contains the insertion site• Insertion of new DNA results in disruption of its

transposase gene• Second P elements has an intact transposase

gene• But it should not itself be transferred, “Wings-clipped’• Its inverted repeats are removed

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Cloning vectors based on insectviruses

Cloning vectors based on insectviruses

• Baculovirus• Play an important role in gene cloning with

other insect• Main use

• Production of recombinant protein• Chapter 13

7.3.2 Cloning in mammals: three reasons

7.3.2 Cloning in mammals: three reasons

• Gene knockout• An important technique used to help determine function of

an unidentified gene• Rodents (mice)

• For production of recombinant protein in amammalian cell culture, pharming• Synthesizes an important protein such as a pharmaceutical• In milk

• Gene therapy• Human cells are engineered in order to treat an disease

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Cloning vectors for mammalsCloning vectors for mammals

• Simian virus 40• 1979• Infecting several

mammalian species• A lytic cycle in some

hosts and lysogeniccycle in other

• 5.2 kb genome

(a) The SV40 genome

Cloning vectors for mammalsCloning vectors for mammals

• A segment of the lategene region wasreplaced

(b) SVGT-5 with insertedrabbit b-globin gene

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Cloning vectors for mammalsCloning vectors for mammals

• Adenoviruses:• Enable large fragment of DNA to be cloned

than SV40• More difficult to handle because genomes are

bigger• Papillomaviruses:

• Relatively high capacity for inserted DNA• Enabling a stable transformed cell lines to be

obtained

Cloning vectors for mammalsCloning vectors for mammals

• Bovine papillomavirus (BPV)• Causes warts on cattle• Take the form of multicopy plasmid with

about 100 molecules per cell• Not cause death of mouse cell• Shuttle vectors: BPV + pBR322

• Retroviruses• At present, most commonly used vector in

mammalian cells• Most important application: gene therapy

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Gene cloning without a vectorGene cloning without a vector

• Microinjection of bacterial plasmids, or linearDNA copies of gene, into mammalian nuclei• Results in DNA being inserted into chromosomes• Possibly as multiple copies in a tandem, head-to-tail

arrangement