Lectut btn-202-ppt-l5. yeast cloning vectors (1)

Biotechnology Engineering

1. Yeast cloning vectors Lecture - 5 1

2. Why is yeast system required? A single cell eukaryote Has short life cycle Genetically well characterized Physiologically well characterized H aploid 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 2

3. Capable of carrying out many post transcriptional and post translational modifications Normally secretes very few proteins Has relatively high rate of recombination 3

4. Transformation of yeast Spheroplasts technique Lithium acetate treatment Electroporation 4

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 5

6. Characteristics of Yeast Cloning V ectors 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 6

7. YEAST INTEGRATING PLASMIDS ( YIps ) Have all features of yeast cloning vectors except origin of replication T hese plasmids therefore replicate only by integration into yeast chromosome E.g.: YIp5 7

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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 9

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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 12

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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 14

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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 16

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 17

18. Y east A rtificial C hromosome 18