A designed cloning vector for two different hosts and selection of transformants

1. By K. K. Gupta

2. Yeast Plasmid
• Plasmids are not limited to bacteria.
• For example, some plasmids have been
extensively studied in yeast and developed
into yeast cloning vectors.
• These plasmids have also been used as
"simple systems" to understand the
mechanism and control of DNA replication in
eukaryotic cells.

3. Introduction
• A shuttle vector is a vector (usually a plasmid) constructed
so that it can propagate in two different host species.
• DNA inserted into a shuttle vector can be tested or
manipulated in two different cell types.
• Also called bifunctional vector since in two host

4. Example
• pHV14,pEB10,pHP3-replicate
in bacillus subtilis & E.coil
• pJDB219-in yeast E coli

5. Features of yeast plasmid
• Yeast has genome about 2X10 7 bp constituting 16 linear
chromosomes,
• Some strains posses a type of plasmid known as the 2 micron circle
containg 6318 bp and in number 70-200 copies
• Yeast hasfour groups depending on replication mode
• YIp- Replicate by integration since lack independent origin for
replication . Yip –one copy /cell – stable , segment of pBR322with ampicilin
+ and bacterial origin
• YRp- Yeast replicating plasmid – has indepent ARS ( autonomus
replicating sequence , but unstable
• YCp- yeast centromeric plasmid- has centromeric sequence that
can attach spindle fibres ( like chromosomes )
• YEp-Yeast episomal plasmids (YEps) are shuttle vectors. They can
replicate in E. coli and also in yeast. Yep can replicate as an
independent plasmid, but also implies that integration into one of
the yeast chromosomes can occur. Number 20-200 /cell
• ... all can be maintained in both bacteria & yeast

6. 2 micron plasmid
• This plasmid is found in several strains of yeast,
Saccharomyces cerevisiae.
• It is 6 kb in size and has a copy number between 70 and 200.
• It replicates autonomously by using several enzymes provided
by the host cell and the proteins coded by its own genes.
• The 2 Micron (µm) Plasmid, 2. LEU2 Gene, and 3. The pBR
322.
• The 2µm plasmid has an origin of replication and two genes
involved in replication, and also encodes a site-specific
recombination protein, called FLP, which is homologous to the
phage λ, integrase and can invert part of the 2µm DNA
sequence

7. 2u circle
• One interesting yeast plasmid is called the 2u circle. The 2u
circle is a 6.3 kb circular, extrachromosomal element found
in the nucleus of most Saccharomyces cerevisiae strains.
• The 2u circle doesn't give cells that carry it any apparent
selective advantage, but it is stably maintained at about 50
to 100 copies per haploid genome of the yeast cells.
• Like the host chromosomes, the 2u circle is coated with
nucleosomes and replication is initiated by host replication
enzymes once per cell cycle.
• The origin of bidirectional DNA replication is initiated at a
specific site on the plasmid called an ARS sequence
("autonomous replication sequence").

8. ARS
• An autonomously replicating sequence (ARS) contains
the origin of replication in the yeast genome. It
contains four regions (A, B1, B2, and B3), named in
order of their effect on plasmid stability. The A-Domain
is highly conserved, any mutation abolishes origin
function.
• Mutations on B1, B2, and B3 will diminish, but not
prevent functioning of the origin.
• Element A is highly conserved, consisting of the
consensus sequence:
• 5'- T/A T T T A Y R T T T T/A -3‘
• Y is either pyrimidine and R is either purine

9. LEU2 gene
• LEU2 gene is a gene of yeast chromosome. It codes for an enzyme
called isopropyl malate dehydrogenase which is involved in
conversion of pyruvic acid to leucine.
• When LEU2 gene is used as a selectable marker, the host yeast
must be a mutant with a non-functional LEU2 gene. Such strains of
yeast are called leu2.
• These mutant cells are unable to synthesize leucine and will grow
only if this amino acid is added to the growth medium.
• However, yeast cells transformed by the YEp (yeast episomal
plasmid) containing LEU2 gene can grow in the growth medium
lacking the amino acid leucine.
• Thus, the transformed yeast cells (called transformants) can be
selected.

10. . The pBR 322
It is the bacterial plasmid. It has two marker genes:
the ampicillin and tetracycline resistant genes (i.e.,
AmpR and TetR). This plasmid has its own origin of
replication.
Hence, initial gene manipulation experiments can be
done with E. coli.
The YEP vector is episomal in nature as it can integrate
with one of the yeast chromosomes. Integration
occurs by homologous recombination between the
LEU2 gene. This results in the insertion of the entire
YEP into on of the yeast chromosome.

11. Shuttle vector construct

12. Genetic Map

14. Advantage
• The main advantage of these vectors is they
can be manipulated in E.coli
• Superior as clone of size 500 kb