Yeast cloning vectors allow DNA fragments to be replicated and expressed in yeast cells. There are several types of yeast vectors including integrating plasmids (YIps) that replicate by integrating into yeast chromosomes, episomal plasmids (YEps) that replicate independently but can also integrate, and replicating plasmids (YRps) that contain an autonomously replicating sequence (ARS) and replicate at low copy numbers. Yeast artificial chromosomes (YACs) are engineered chromosomes containing telomeric, centromeric, and ARS sequences that can clone very large DNA fragments of up to 3000 kb.

1. Yeast cloning vectors
Lecture - 5
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2. Why is yeast system required?
•A single cell eukaryote
•Has short life cycle
•Genetically well characterized
•Physiologically well characterized
•Haploid genome has low complexity with size of nearly 12
Mbp
•Easy to grow
•Many auxotrophic and other markers are known
• Several strains harbor 2 µm plasmid
•A source of several strong promoters
•Introduction of naked DNA is easy
•Many yeast genes are functionally expressed in E.coli
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3. •Capable of carrying out many post transcriptional and
post translational modifications
•Normally secretes very few proteins
•Has relatively high rate of recombination
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4. Transformation of yeast
1.Spheroplasts technique
2.Lithium acetate treatment
3.Electroporation
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5. Yeast 2 µm Plasmid
•Covalently closed
circular DNA
molecule
•Consists of 6318bp
of double stranded
DNA
•50-100 copies per
haploid cell
•Located in nucleus
of yeast cell
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6. Characteristics of Yeast Cloning Vectors
•Presence of both E. coli and yeast origins
of replication
•Presence of unique restriction enzyme
sites for cloning
•Presence of selectable markers for both E.
coli and yeast
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7. YEAST INTEGRATING PLASMIDS (YIps)
•Have all features of yeast cloning vectors
except origin of replication
•These plasmids therefore replicate only by
integration into yeast chromosome
•E.g.: YIp5
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9. YEAST EPISOMAL PLASMIDS (YEps)
•YEps replicate independently but
integration into one of the yeast
chromosomes can occur
• YEps are designed as double shuttle
vectors
• E.g.: YEp13
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12. YEAST REPLICATING PLASMIDS (YRps)
•YRps are ARS ( Autonomously Replicating Sequence) -
based vectors
•Exist as low copy number
•Have high transformation efficiency
•Have natural extra chromosomal replication due to
ARS
•YRps tend to remain associated with mother yeast
cells and have less chances of distribution to daughter
cells
•E.g. : YRp7
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14. YEAST CENTROMERIC PLASMIDS (YCps)
•Autonomously replicating
•YCps transform yeast at high efficiency but
transformants are unstable
•E.g.: YCp 50
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16. YEAST ARTIFICIAL CHROMOSOMES
•A yeast artificial chromosome (YAC) is a vector used
to clone DNA fragments larger than 100 kb and up to 3000
kb
•YACs are useful for the physical mapping of complex
genomes and cloning of large genes
• A YAC is an artificially constructed chromosome and
contains the telomeric, centromeric, and replication origin
sequences named autonomous replicating sequence
(ARS) needed for replication in yeast cells
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17. 1987
Construction of Yeast
Artificial Chromosome
(YAC)
Cloning of large segments of
exogenous DNA into yeast by means
of artificial chromosome vectors
250-400 kb
810 kb
1800 kb
Washington University, USA
Maynard Olson
Gruber Prize for Genetics 2007
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18. Yeast Artificial Chromosome
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