Yeast Vectors

Yip Vectors The Ypl integrative vectors do not replicate autonomously, but integrate into the genome at low frequencies by homologous recombination. Integration of circular plasmid DNA by homologous recombination leads to a copy of the vector sequence flanked by two direct copies of the yeast sequence. 

YEp Vectors The YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2 pm plasmid that serves as an origin of replication (2 pm oii). The 2 pm ori is responsible for the high copy-number and high frequency of transformation of YEp vectors. 

YCp Vectors The YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences, CEN, and autonomously repheating sequences, ARS. 

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Steinborn, G., Boer, E., Scholz, A. et al. (2006) Application of a wide-range yeast vector (CoMed) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts. Microbial Cell Factories, 5, 33. 

In 1993, Kikuta et cloned a P450 now known as P450 4F3 from a human leukocyte cDNA library. The protein was expressed in a yeast vector system and was shown to catalyze leukotriene B w-hydroxylation. The (0.7 p,M) was much lower than that reported for P450 4F2 for this reaction (although the and k K values have not been carefully compared) . ... 

Mumberg, D., Muller, R., and Funk, M. (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene,... 

Starmer, W.T., Phaff, H.J., Bowles, J.M., Lachance, M.-A. (1988b). Yeasts vectored by insects feeding on decaying saguaro cactus. Southwestern Naturalist 33, 362-363. 

Reynolds, A. and Lundblad, V (1989) Yeast vectors and assays for expression of cloned genes, in Current Protocols in Molecular Biology (Ausubel, F A, Brent, R., Kingston, R E., Moore, D D, Seidman, J. G., Smith, J A, and Struhl, K., eds ), Greene Publishing and Wiley-Interscience, pp 13 6 1-13 6 4. 

Eig. 5. Restriction map of the yeast artificial chromosome (YAC) vector used for cloning very large fragments of eukaryotic DNA. Terms defined in text... 

A variety of tiansformation techniques using E. co/ -ye st shuttle vectors and yeast selectable markets, as well as efficient yeast promoters and signal... 

Yeast (Saeeharomyees eerevisiae) has a genome size of 1.21 X 10 bp. If a genomic library of yeast DNA was constructed in a bacteriophage A vector capable of carrying 16-kbp inserts, how many indi-... 

In general, fungal mycelia are filtered relatively easily, because mycelia filter cake has sufficiently large porosity. Yeast and bacteiia are much more difficult to handle because of thefr small size. Alternative filtration methods, which eliminate the filter cake, are becoming more acceptable for bacterial and yeast separation. Micro-filtration is achieved by developing large cross-flow fluid velocities across the filter surface while the velocity vector normal to the surface is relatively small. Build up of filter cake and problems of high cake resistance are therefore prevented. Micro-filtration is not discussed in this section. 

A Bacterial Artificial Chromosome (BAC) is a vector that allows the propagation of larger exogenous DNA fragments, up to several hundred kb. BACs are propagated in recombination-deficient strains of E. coli. They are more stable and easier to handle than yeast artificial chromosomes (YACs). 

An yeast artificial chromosome (YAC) is a vector that allows the propagation of large exogenous DNA fragments, up to several megabases, in yeast. 

Each of the -6000 PCR products was then co-transformed into yeast along with the recipient vector that had been linearized using a restriction enzyme that digests the plasmid at the desired cloning site. The 70 bp of homologous flanking sequence on each end of the PCR products is sufficient for the yeast homologous recombination system to act upon and insert the PCR product into the vector (Hudson et al., 1997 Ma et al., 1987). 

Figure 4.7. In vivo recombinational cloning in yeast. Two successive PCR reactions are performed. Each set of primers contains 5 flanking sequences that are eventually used for homologous recombination with vector sequences after transformation of yeast with the 2nd PCR fragment and the linearized vector.

Figure 7.6. Purification of protein from pooled yeast strains. Each yeast ORF was cloned as a fusion to glutathione-S-transferase in a protein expression vector to create 6144 yeast strains. The individual strains were pooled in groups of 96 to create a set of 64 pools. Each pool was grown and the 96 fusion proteins are purified in batch. Each pool was then assayed for a biochemical function (Martzen et al., 1999). Pools positive for function were then deconvoluted using smaller pools consisting of strains from rows and columns of a 96-well plate.

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