SHUTTLE VECTORA.NEERAJA

VECTOR - DEFINITION

A vector is an organism that does not cause disease itself but which spreads

infection by conveying pathogens from one host to another.

DNA molecule originating from a virus, a plasmid, or the cell of a higher organism

into which another DNA fragment of appropriate size can be integrated without the

loss of the vector capacity for self-replication.

Vectors introduce foreign DNA into the host cells, where it can be reproduced in

large quantities.

REASON FOR DEVELOPING SHUTTLE VECTOR

Prokaryotic vectors cannot exist & work in eukaryotic cells because the system of

two groups of organisms varies.

Prokaryotes lack introns; while eukaryotes consists of introns.

Therefore, vectors with two origin of replication were constructed which may exist

in both eukaryotes and prokaryotes.

SHUTTLE VECTOR - DEFINITION

A vector usually a plasmid constructed so that it can propogate in two different

host species.

Since these vectors can be grown in one host and then moved into another without any extra manipulation, they are called shuttle vectors.

These vectors have been designed to replicate in cells of two different species; therefore, they contain two origins of replication, one specific for each host species, as well as those genes necessary for their replication and not provided by the host cells.

•These vectors are created by recombinant techniques.

•Some of them can be grown in two different prokaryotic species, usually E. coli and a eukaryotic one, e.g. yeast, plants, animals.

YEp13 is an example of shuttle vector.

•The yeast Saccharomyces cerevisiae is one of the most important organisms inbiotechnology.

Development of cloning vectors for yeast was initially stimulated by the discovery of a plasmid that is present in most strains of S. cerevisiae.

The 2 µm plasmid, as it is called, is one of only a very limited number of plasmids found in eukaryotic cells.

Vectors derived from the 2 µm plasmid are called yeast episomal plasmids (YEps).

Some YEps contain the entire 2 µm plasmid, others include just the 2 µm origin of

replication. An example of the latter type is YEp13.

The 2 µm plasmid is an excellent basis for a cloning vector. It is 6 kb in size, which

is ideal for a vector, and exists in the yeast cell at a copy number of between 70

and 200. Replication makes use of a plasmid origin, several enzymes provided by

the host cell, and the proteins coded by the REP1 and REP2 genes carried by the

plasmid.

However, all is not perfectly straightforward in using the 2 µm plasmid as a cloning vector. First, there is the question of a selectable marker.

Here gene LEU2, which codes for b-isopropyl-malate dehydrogenase, one of the enzymes involved in the conversion of pyruvic acid to leucine is used as marker.

In order to use LEU2 as a selectable marker, a special kind of host organism isneeded. The host must be an auxotrophic mutant that has a non-functional LEU2 gene.

•Such a leu2− yeast is unable to synthesize leucine and can survive only if this amino acid is supplied as a nutrient in the growth medium. Selection is possiblebecause transformants contain a plasmid-borne copy of the LEU2 gene, and so are able to grow in the absence

of the amino acid.

In a cloning experiment, cells are plated out onto minimal medium, which contains no added amino acids. Only transformed cells are able to survive and form colonies.

YEp13 illustrates several general features of yeast cloning vectors. First, it is a shuttle vector.

As well as the 2 µm origin of replication and the selectable LEU2 gene, YEp13 also includes the entire pBR322 sequence, and can therefore replicate and be selected for in both yeast and E. coli.

USES OF SHUTTLE VECTOR

it might be difficult to recover the recombinant DNA molecule from a transformed yeast colony.

This is not such a problem with YEps, which are present in yeast cells primarily as plasmids, but with other yeast vectors, which may integrate into one of the yeast chromosomes, purification might be impossible.

This is a disadvantage because in many cloning experiments purification of recombinant DNA is essential in order for the correct construct to be identified by, for example, DNA sequencing.

The standard procedure when cloning in yeast is therefore to perform the initialcloning experiment with E. coli, and to select recombinants in this organism.Recombinant plasmids can then be purified, characterized, and the correct molecule introduced into yeast.

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