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YEAST SHUTTLE VECTORS
BIOTECHNOLOGY
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YEAST SHUTTLE VECTORS

A shuttle vector is a vector constructed in such a way that they have the origins of
replication two different host species, either eukaryotes or prokaryotes or simply in
different species of bacteria. They are frequently used to quickly make multiple copies
of the gene in E. coli, manipulate it and then use it in a system which is more difficult or
slower (e.g. yeast, other bacteria). They can also be used for in vitro experiments and
modifications (e.g. mutagenesis, PCR)

Yeast is one of the most common types of shuttle vectors. Although there are
numerous kinds of yeast shuttle vectors, those which are currently use can be broadly
classified in the following types:

 YIp Vectors: integrative vectors

 Do not replicate autonomously

 Stable in the yeast only on integration into the yeast chromosome.

 Typically integrate as a single copy

 Multiple integration occurs at low frequencies

 Integration of circular plasmid DNA leads by homologous recombination to

a copy of the vector sequence flanked by two direct copies of the yeast
sequence

 Restriction endonuclease is used to cut the yeast segment at the site of

integration, transforming the yeast strain with the linearized plasmid

 Linearization increases the efficiency of integrative transformation from

10- to 50-fold.

 Strains constructed with YIp plasmids should be examined by PCR

analysis, or other methods, to confirm the site of integration.

 YEp - Yeast episomal plasmids:

  Carry 2 mm circles of DNA sequence including the origin of replication

 Replicate autonomously

 This origin of replication is responsible for the high copy-number and high
frequency of transformation of YEp vectors.

 Most YEp plasmids are relatively unstable, being lost in approximately 10-
2 or more cells after each generation.

 Stable only in dividing cells

 Even under conditions of selective growth, only 60% to 95% of the cells
retain the YEp plasmid.

 The plasmids are not equally distributed among the cells, and there is a
high variance in the copy number per cell in populations.

 The most common use for YEp plasmid vectors is to overproduce gene
products in yeast

 Example is YEp13.

 YCp Vectors- yeast centromere plasmid

 Function as true chromosomes and segregate during mitosis and meiosis.

 Containing centromere sequences, CEN, and autonomously replicating

sequences, ARS.

 Typically present at very low copy numbers, from 1 to 3 per cell, and
possibly more.

 Lost in approximately 10-2 cells per generation without selective pressure

 Segregate to two of the four ascospore from an ascus, indicating that they
mimic the behavior of chromosomes during meiosis, as well as during
mitosis.

 The ARS sequences are believed to correspond to the natural replication

origins of yeast chromosomes, and all of them contain a specific
consensus sequence
  The CEN function is dependent on three conserved domains, namely I, II,
and III; all three of these elements are required for mitotic stabilization of
YCp vectors

 The stability and low copy-number vectors make them the best choice for
cloning vectors, for construction of yeast genomic DNA libraries, and for
investigating the function of genes altered in vivo.

 Example: YCp50 containing CEN4 and ARS1.

 YAC - Yeast artificial chromosome vector:

 Carries centromere and telomere sequences and therefore can be used to

obtain artificial chromosomes

 Used for cloning large fragments

 Linear chromosomes, very stably maintained

 Useful for accepting very long range physical mapping techniques, used in
human genome analysis

 For cloning, the vector is first restricted with a combination of Bam HI and
Sna BI, cutting the molecule into three fragments

 The DNA to be cloned, which must have blunt ends (Sna BI, a blunt end
cutter, recognizes TACGTA) is ligated between the two arms producing
the artificial chromosome

 Example: pYAC3, which is essentially a pBR 322 into which a number of

yeast genes have been inserted.

REFERENCES:
1. http://books.google.com.pk/books?
id=8rR6Prg3TcC&pg=PA275&lpg=PA274&ots=zxflokbc2r&dq=yip+yeast+integrative+plasmid#v=
onepage&q=yip%20yeast%20integrative%20plasmid&f=false
2. http://www.studentsguide.in/animal-biotechnology/gene-cloning/YIp-yest-integrative-
plasmid.html
3. http://www.studentsguide.in/animal-biotechnology/gene-cloning/YEp-yeast-episomal-
plasmids.html
4. http://www.studentsguide.in/animal-biotechnology/gene-cloning/pYAC-yeast-artificial-
chromosome-vector.html
5. http://en.wikipedia.org/wiki/Shuttle_vector