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 tended to 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 most common 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 the United States, Canada, the United Kingdom, and Japan. “In 1993, we made a gentleman's' agreement not 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 Union initiative from the Catholic University of Louvain in Belgium. "We agreed not to stake out any territory and, on several occassions, DNA fragments to be sequenced were redistributed according to the respective abilities of the sequencing teams.” 1, 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 compound required 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 unable to synthesise an essential compound. For example a yeast mutant in which a gene of the uracil synthesis pathway is inactivated is a uracil auxotroph. Such a strain is unable to synthesise 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, which can still grow in the absence of uracil. Auxotrophic genetic markers are often used in molecular genetics.

pESC-HIS The pESC vectors are a series of epitope-tagging vectors designed for expression and functional analysis of eukaryotic genes in the yeast S. cerevisiae. Each vector contains the GAL1 and GAL10 yeast promoters in opposing orientation With these vectors one or two cloned genes can be introduced into a yeast host strain under the control of a repressible promoter. When two genes are co-expressed, protein-protein interactions can be confirmed by immunoprecipitation analysis. These vectors feature an extensive polylinker sequence and the ability to generate end-specific RNA transcripts from T3 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-fol in cells growing on galactose and is repressed by growth on glucose. (Mol Cell Biol August; 4(8): 1440–1448.) In pESC vectors (Stratagen) both the GAL1 and GAL10 promoters from S. cerevisiae are strictly regulated at the transcription level by the carbon source in the media. These promoters are tightly repressed when glucose is present in the media and are highly induced when galactose is the sole carbon source. In S. cerevisiae, the induction ratio of these promoters has been estimated to be greater than 1000 fold. The presence of both the GAL1 and GAL10 promoters in opposite orientatio allows two genes to coexpress in the same host cell.

Epitope Tagging The pESC vectors contain DNA sequences coding for epitope peptides that can be specifically 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 GAL1 promoter. The gene of interest can be inserted in front of the epitope sequence to generate C-terminal tagging or after the epitope sequence for N-terminal tagging. These tags allow the protein of interest to be studied without generating a specific antibody to that protein. The epitope-tagged fusion proteins can be studied in transformed cells using well-characterized antibodies

pYES vectors the 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 region CYC TT: CYC teranscription termination. V5 epitope tag: GKPIPNPLLGLDST

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

PYES-DEST52 The pYES-DEST52 vecto combines the Gateway technology with the regulated expression of the pYES2 vector.

pYC2/NT

pPICZA,B,C

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