Saccharomyces cerevisiae :

Saccharomyces cerevisiae is commonly known as "bakers yeast" or "brewers yeast".The yeast ferments sugars present in the flour or added to the dough,giving off carbon dioxide (CO2) and alcohol (ethanol).The CO2 is trapped as tiny bubbles in the dough, which rises.

Before the use of Saccharomyces cerevisiae, bread was tough, dry stuff that tendedto break your teeth and made your jaw ache.

Bread made with yeast was wonderful, light, tasty stuff.

It is the most intensively studied eukaryotic model organisms in molecular and cell biology,much like Escherichia coli as the model prokaryote. It is the microorganism behind the mostcommon type of fermentation.

Saccharomyces cerevisiae cells are round to ovoid, 5-10 Micrometres in diameter.It reproduces by a division process known as budding.

The yeast sequencing initiative involved 92 laboratories the European Union, as well as labs in theUnited States, Canada, the United Kingdom, and Japan. “In 1993, we made a gentleman's' agreementnot to compete, but to divide the work among us in order to complete the sequence rapidly with as little duplication as possible," said Dr. Andre Goffeau, who coordinated the European Unioninitiative from the Catholic University of Louvain in Belgium."We agreed not to stake out any territory and, on several occassions, DNA fragments to be sequencedwere redistributed according to the respective abilities of the sequencing teams.”1,4 10 7 nucleotides

Auxotroph:

is a mutant organism requiring a specific growth substance not normally required by its species.

Auxotrophy is the inability of an organism to synthesize a particular organic compoundrequired for its growth. An auxotroph is an organism that displays this characteristic;auxotrophic is the corresponding adjective. Auxotrophy is the opposite of prototrophy.

In genetics, a strain is said to be auxotrophic if it carries a mutation that renders it unableto synthesise an essential compound. For example a yeast mutant in which a gene of theuracil synthesis pathway is inactivated is a uracil auxotroph. Such a strain is unable tosynthesise uracil and will only be able to grow if uracil can be taken up from the environment.

This is the opposite of a uracil prototroph, or in this case a wild-type strain, whichcan still grow in the absence of uracil. Auxotrophic genetic markers are often usedin molecular genetics.

pESC-HISThe pESC vectors are a series of epitope-tagging vectors designed for expressionand functional analysis of eukaryotic genes in the yeast S. cerevisiae.Each vector contains the GAL1 and GAL10 yeast promoters in opposing orientationWith these vectors one or two cloned genes can be introduced into a yeast host strainunder the control of a repressible promoter.When two genes are co-expressed, protein-protein interactions can be confirmed byimmunoprecipitation analysis. These vectors feature an extensive polylinker sequenceand the ability to generate end-specific RNA transcripts fromT3 and T7 promoters.Each of the pESC vectors contains one of four different yeast-selectable markers(HIS3, TRP1, LEU2, or URA3) in the same vector backbone.

GALl1 and GAL 10 promoters (PGAL1 and PGAL10)

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed,with 606 base pairs of DNA separating their transcription initiation sites.These two genes are stringently coregulated: their expression is induced ca. 1,000-folin cells growing on galactose and is repressed by growth on glucose.(Mol Cell Biol. 1984 August; 4(8): 1440–1448.)

In pESC vectors (Stratagen) both the GAL1 and GAL10 promoters from S. cerevisiae arestrictly regulated at the transcription level by the carbon source in the media. These promoters are tightly repressed when glucose is present in the media andare highly induced when galactose is the sole carbon source. In S. cerevisiae, the inductionratio of these promoters has been estimated to be greater than 1000 fold.The presence of both the GAL1 and GAL10 promoters in opposite orientatioallows two genes to coexpress in the same host cell.

Epitope Tagging

The pESC vectors contain DNA sequences coding for epitope peptides that can bespecifically recognized by monoclonal antibodies.A sequence coding for DYKDDDDK is located in MCS downstream of the GAL10 promoter;a sequence for the c-myc epitope EQKLISEEDL is located in the MCS downstream of the GAL1promoter.The gene of interest can be inserted in front of the epitope sequence to generate C-terminaltagging or after the epitope sequence for N-terminal tagging.These tags allow the protein of interest to be studied without generating a specificantibody to that protein. The epitope-tagged fusion proteins can be studied in transformedcells using well-characterized antibodies

pYES vectorsthe pUC family of vectors are high copy vectors.They have a ColE1 origin of replication, but a deletion of the rop replication regulatory region

the pUC family of vectors are high copy vectors.They have a ColE1 origin of replication, but a deletion of the rop replication regulatory regionCYC TT: CYC teranscription termination.V5 epitope tag: GKPIPNPLLGLDST

pYES2.1/V5-His-TOPOpYES2.1/V5-His-TOPO show the TOPO Cloning,With this vector, you can clone anyr Taq amplifiedPCR products in a 5-minute benchtop incubation

PYES-DEST52The pYES-DEST52 vecto combines the Gateway technology with the regulated expression ofthe pYES2 vector.

pYC2/NT

pPICZA,B,C

Example:

FIG. 1. (a) pGAPZa A expression construct. PGAP, GAP promoter region. AOX1 TT, alcohol oxidase transcriptiontermination region.PTEF1, promoter region of transcription elongation factor 1. PEM7,promoter conferring Zeocinresistance. Zeocin, a selectable marker. CYC1 TT, CYC1 transcription termination region. ColE1 origin allowingreplication and maintenance of the plasmid in E. coli. (b) Construction of the two recombinant hAFPs. WT andWT-6H were designed to express mature hAFP and mature hAFP with a six-histidine tag, respectively.