Yeast molecular biology: application

Presented by:- sonu m.sc mbt 2nd sem roll no. 1533

content

• Introduction• Important yeast• Yeast as a model organism• Cloning in the yeast• Yeast E.coli shuttle vector• Yeast as expression vector• Yeast two hybrid system• Application of yeast two hybrid system• Conclusion• Refrence

Introduction

• Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with 1,500 species currently described.

• Mostly unicellular, some species may become multicellular.• Size is about 3–4 µm in diameter.• Reproduce asexually by mitosis, and by an asymmetric division

process called budding.• Also known as chemoorganotrophs.• Often isolated from sugar-rich materials such as on the skins of

fruits and berries.•  Some are found in association with soil and insects.

Pichia postoris

Important yeast

• S. cerevisiae is the main organism in wine production besides other yeasts; because of its : – enormous fermentation capacity– low pH and high ethanol tolerance.

• It is the beer yeast as it ferments sugar to alcohol even in the presence of oxygen.

• Used in baking because it produces CO2 from sugar very rapidly.• Used to produce commercially important proteins.• Also used for drug screening and functional analysis because it is a

eukaryote but can be handled as easily as bacteria.• One of the most important eukaryotic cellular model system.

Other important yeasts

• Schizosaccharomyces pombe the fission yeast; important model organisms in molecular and cellular biology; used for certain fermentations

• Candida albicans not a good model since it lacks a sexual cycle; but studied intensively because it is human pathogen.

• Pichia stipidis, Hansenula polymorpha, Yarrovia lipolytica have smaller importance for genetic studies , protein production hosts.

Yeast as model organisms• S. cerevisiae and S. pombe are regarded as model organisms in molecular

biology.• Most of the principal cellular systems function in a similar way in yeasts and

human, i.e. across eukaryotes.• But yeasts are not just simple human cells.• Yeasts are unicellular and hence lack an important level of complexity.• By the way, although S. cerevisiae and S. pombe are both yeasts, they are as

distinct from each other than each is from human

S. cerevisiae S. pombe

Human

Model character: morphology switch

• Yeast cells can switch their morphology i.e. Filamentous or unicellular.

• The yeast pseudohyphal switch is a model system for morphogenesis.• Most importantly, a morphological switch is associated with

pathogenesis of C. albicans and C. neoformans.• S. cerevisiae may use the switch and co-expression of polysaccharide

degrading enzymes to penetrate plant tissues.

Model character: control of gene expression

• Control of gene expression means that signals and molecules have to traverse the nuclear membrane and these mechanisms seem to be well conserved.

• The principles of the control of transcription are well conserved across eukaryotes and many proteins function across species eg. transcription factors.

• The organisation of the transcription initiation machinery seems to be conserved.

• The mechanisms of transcriptional activation seem to be conserved, but certain classes of activators (proline- and glutamine-rich) do not seem to function in yeast.

• Chromatin organisation is more simple in yeast, aspects of its involvement in the control of gene expression are similar.

Model character: vesicular transport

• Vesicular transport, i.e. the mechanisms that control the trafficking of proteins is another feature that is highly conserved across eukaryotes

• Temperature sensitive sec mutants have been sorted according to the stage where transport stops (using electron micoscopy) and this has been the foundation for genetic analysis.

• In addition, transport to the vacuole and endocytosis are studied by genetic analysis.

Model character: Proteasome• The proteasome is a multi protein complex conserved in eukaryotes.• It is located in the cytoplasm and the nucleus and controls degradtion of proteins

that have been ubiquitinated.• The 26S proteasome consist of a 20S catalytic and a 19/22S regulatory subunit• The 20S proteasome is composed of 14 different proteins and all genes are known

in yeast• The yeast 20S complex has been purified and the X-ray structure has been

determined

Cloning in yeast• The era of yeast molecular genetics started as early as 1978, when S.

cerevisiae was first transformed successfully with foreign DNA.• There are numerous transformation protocols but all are at least

three orders of magnitude less efficient as transformation in E. coli.• Yeast can maintain replicating plasmids but the copy number is

much smaller than in E. coli, usually between one and 50 per cell.• Yeast can maintain more than one type of plasmid at the same time.

This can complicate gene cloning from a library. • Cloning and plasmid preparation from yeast is very ineffective.• Therefore, cloning in yeast uses E. coli as a plasmid production

system, so-called yeast-E. coli shuttle vectors

Yeast-E. coli shuttle vectors

• Yeast Integrative plasmid (YIp) consist:– backbone of a E. coli vector such as pBR322, pUC19,

pBLUESCRIPT.– Has yeast selection marker such as URA3, HIS3, TRP1,

LEU2.• They are lacking any replication origin for yeast• Hence, they are propagated only through integration into the

genome

Replicative episomal plasmids (YEp)

• Consist backbone of a E. coli vector such as pBR322, pUC19, pBLUESCRIPT– a yeast selection marker such URA3, HIS3, TRP1, LEU2

• Have the replication origin of the yeast 2micron plasmid, so they can replicate indipendently.

• High copy number, typically 20-50 per cell.• Their copy number can be pushed to 200 per cell by using as

marker a partially defective LEU2 gene.

Yeast as expression vectors• The production of proteins is of interest for several purposes:

– For research : purification and structural analysis– For industry :production of enzymes for the food and paper industry or for

research and diagnostics– For pharmaceutical industry for the production of vaccines

• There are a number of different expression hosts, such as bacteria and yeasts.• Yeast have the advantage that they may perform the same or at least similar post-

translation modifications, such as glycosylation• Yeast usually reaches only a lower level of expression.• The apparently most productive known yeast is the species P. pastoris; it

catabolises methanol and the promoter for methanol oxidase is extremely strong and can be induced by methanol.

• In S. cerevisiae one usually uses the promoters of genes encoding glycolytic enzymes such as PGK1 and TPI1 or a regulated promoter such as that of GAL1

• The advantage of S. cerevisiae is that so much is known about its molecular biology and one can device genetic screens to improve protein production and secretion

Yeast two-hybrid system• The yeast two hybrid system is a method to detect the interaction of two proteins in the

yeast cell and it can be used to select for an interacting partner of a known protein• The original version uses a transcriptional read-out to monitor interaction.• The method is so powerful since it is not restricted to yeast proteins; the interacting

partners can origin from any organism; in fact some versions do not use any yeast sequences.

• Basis for the system is the modular nature of transcription activators that consist of exchangeable DNA binding and transcriptional activation domains.

lexAsite

reporter

The gene of interest, the bait, is cloned in fusion with a DNA binding domain, such as that of the E. coli lexA protein

The potential binding partner or prey, which may be a library, is cloned in fusion to a transcriptional activation domain, such as that from VP16, a viral protein

Only when bait and target interact, a reporter gene whose only promoter is a lexA binding site will be activated

Application of the yeast two-hybrid system

• Used to detect interaction between two proteins.• Used to characterise the domains and residues in the two proteins

that mediate interaction.• Used to find interaction partners.• Used to find proteins that regulate the interaction between two

proteins.• Used to screen for drugs that inhibit the interaction between two

proteins.• Used to construct a genome-wide map of protein interactions in

yeast.

Conclusion• Yeast are the important class of microorganisms.• It is also a centrally important model organism in

modern cell biology research, and is one of the most thoroughly researched eukaryotic microorganisms.

• Researchers have used it to gather information about the biology of the eukaryotic cell and ultimately human biology.

• In industries they have been used in production of wine, beer, bread and other beverages and fermented food.

• Many yeast are also pathogens which cause infectious diseases.

Refrence

• Strathern, J. N., Jones, E. W., and Broach, J. R. (1982). The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

• Beggs, J. D. Transformation of yeast by a replicating hybrid plasmid. Nature 275, 104–109 (1978).

• Sipiczki, M. Phylogenesis of fission yeasts — contradictions surrounding the origin of a century old genus. Antonie Van Leeuwenhoek 68, 119–149 (1995).

• Grallert, B., Nurse, P. & Patterson, T. E. A study of integrative transformation in Schizosaccharomyces pombe. Mol. Gen. Genet. 238, 26–32 (1993).

• Rine, J. Gene overexpression in studies of Saccharomyces cerevisiae. Methods Enzymol. 194, 239–251 (1991).

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