Saccharomyces cerevisiae

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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.1,4 10 7 nucleotides
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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 aux
otrophic 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.
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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.
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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.
1984 August 4(8) 14401448.) 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.
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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
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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
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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
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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
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PYES-DEST52
The pYES-DEST52 vecto combines the Gateway
technology with the regulated expression of the
pYES2 vector.
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pYC2/NT
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pPICZA,B,C
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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.
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