yeast as a model eukaryotic organism - tcd

Yeast Genetics

• The yeast genome can be manipulated.

• Can introduce genes into yeast cells by

transformation.

• Can delete genes.

• Can mutate genes: look for altered

phenotypes.

Yeast genetics: nomenclature

• Yeast genes have names consisting of three letters and up to three numbers:ACT1, HSP12, PDC6...Usually they are meaningful (or meaningless) abbreviations

• Wild type genes are written with capital letters in italics: TPS1, RHO1,CDC28...

• Recessive mutant genes are written with small letters in italics: tps1, rho1, cdc28

• Mutant alleles are designated with a dash and a number: tps1-1, rho1-23, cdc28-2

• If the mutation has been constructed, i.e. by gene deletion, this is indicated and the genetic marker used for deletion too: tps1D::HIS3

• The gene product, a protein, is written with a capital letter at the beginning and not in italics; often a ”p” is added at the end: Tps1p, Rho1p, Cdc28p

Yeast genetics: nomenclature

• Many genes have of course only be found by systematic sequencing and as long as their function is not determined they get a landmark name: YDR518C, YML016W..., where

– Y stands for ”yeast”

– The second letter represents the chromosome (D=IV, M=XIII....)

– L or R stand for left or right chromosome arm

– The three-digit number stands for the ORF counted from the centromere on that chromosome arm

– C or W stand for ”Crick” or ”Watson”, i.e. indicate the strand or direction of the ORF

• Some genes do not follow this nomenclature: you heard already about: HO, MATa, MATa

Yeast genetics: markers and

strains• Genetic markers are used to follow chromosomes in crosses

and in transformation of plasmids

• Commonly genetic markers cause auxotrophies: HIS3, URA3, TRP1, LEU2, LYS2, ADE2

• The ade2 mutation has a specific useful feature: cells turn red

• Like in E. coli also certain antibiotic resistance markers can be used in transformation: kanamycin resistance, kanR

• There are many yeast strains in use in the laboratories: W303-1A, S288C, S1278b, SK1, BY4741....

• Their specific properties can be quite different and are different to wild or industrial strains

• The full genotype of our favourite strain W303-1A reads like this:

MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0

• Plasmids can be

used to introduce

genes into yeast.

• Use auxotrophic

markers to follow

introduction of

plasmid with new

trait.

• Plate cells on ura

minus plates

Amp-resistance

Tet-resistance

YCp507950bp URA3

CEN4

ARS1

PMB1

MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1

can1-100 GAL SUC2 mal0

• Auxotrophic mutants such as ura will not grow

on minimal media plates lacking uracil.

• Auxotrophic mutants will grow on minimal

media plates if all essentail nutrients are

provided.

Auxotrphic mutants containing a plasmid with a

URA gene will grow on minimal media plates

minus uracil

Yeast genetics: markers and

strains• Genetic markers are used to follow chromosomes in crosses

and in transformation of plasmids

• Commonly genetic markers cause auxotrophies: HIS3, URA3, TRP1, LEU2, LYS2, ADE2

• The ade2 mutation has a specific useful feature: cells turn red

• Like in E. coli also certain antibiotic resistance markers can be used in transformation: kanamycin resistance, kanR

• There are many yeast strains in use in the laboratories: W303-1A, S288C, S1278b, SK1, BY4741....

• Their specific properties can be quite different and are different to wild or industrial strains

• The full genotype of our favourite strain W303-1A reads like this:

MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0

Yeast genetics: the genetic

material

• The S. cerevisiae nuclear genome has 16 chromosomes

• In addition, there is a mitochondrial genome and a plasmid, the 2micron circle

• The yeast chromosomes contain centromeres and telomeres, which are simpler than those of higher eukaryotes

• The haploid yeast genome consists of about 12,500 kb and was completely sequenced as early 1996 (first complete genome sequence of a eukaryote)

Yeast genetics: the

genetic material

• The yeast genome is predicted to contain about 6,200 genes, annotation is, however, still ongoing. New data suggests there are approximately 5,500 ORFs.

• Roughly 1/3 of the genes has been characterised by genetic analysis, 1/3 shows homology hinting at their biochemical function and 1/3 is not homologous to other genes or only to other uncharacterised genes

Yeast genome

analysis

• A joint goal of the yeast research community: determination of the function of each and every gene

• For this, there are several large projects and numerous approaches

• Micro array analysis: simultaneous determination of the expression of all genes

• Micro array analysis to determine the binding sites in the genome for all transcription factors

• Yeast deletion analysis: a complete set of more than 6,000 deletion mutants is available for research

Yeast genome analysis

• Yeast deletion analysis: a complete set of more than 6,000 deletion mutants is available for research. Genes deleted singly and now starting to delete in pairwise fashion.

• Various approaches to analyse the properties of these mutants, e.g are genes essential for viability, growth conditions etc.

• All yeast genes have been tagged to green fluorescent protein (GFP) to allow protein detection and microscopic localisation

• Proteomic analysis: Identify all proteins present in the cell under a specific set of conditions.

• Different global protein interaction projects are ongoing: identify all proteins that interact with each other.

Ultimate analysis

• Complete sequence of genome

• Analysis of expression of all genes (Transcription) under as many conditions as possible. (Transcriptome)

• Analysis of all proteins produced (Translation) under as many conditions as possible.(Proteome)

• Protein-Protein interactions.

• Analysis of all metabolic reactions taking place under specific conditions (Metabolome)

Yeast as a model Eukaryotic

Organism

The Saccharomyces Genome

Database

http://www.yeastgenome.org/

• A model database for the analysis of

eukaryotic genomes.

The Saccharomyces Genome

Database• ORFs ordered along each chromosome and

annotated YPR103W.

• SGD contains data on each ORF such as DNA sequence, protein encoded by gene,

• Biological function, transcription profile under a variety of environmental conditions.

• Links to other important sites such as protein analyis, protein-protein interactions,

• Links to NCBI for nucleotide and amino acid comparisons to genes/proteins in other species.

The Saccharomyces Genome

Database

• A model system for other organisms

such as the human DNA sequencing

project.

Standard Name HTB1Click on map for expanded view

* LiteratureView

* Retrieve SequencesRetrieve

* Sequence Analysis ToolsAnalyze

* Protein Info & StructureView

* Localization ResourcesView

* InteractionsView

* Maps and DisplaysView

* Comparison ResourcesView

* Functional AnalysisView

Alias SPT12Systematic Name YDR224CFeature Type ORF, VerifiedDescription Histone H2B (HTB1 and HTB2 code for

nearly identical proteins)GO Annotations

Molecular FunctionBiological ProcessCellular Component

The model organisms

• The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are regarded as model organisms in molecular biology

• Extrapolate to humans

• But yeasts are not just simple human cells

• yeasts are unicellular and hence lack an important level of complexity, i.e. that of a multicellular organism

• 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: eukaryotic

cell cycle• Learn about cell cycle control

• Nutrient starvation and pheromone cause cell cycle arrest at this point

• Identified mutants that disturb cell cycle

The actin cytoskeleton

during the cell cycle

Model character: signal

transduction• common classes of signalling proteins, such as G-protein coupled receptors, the

yeast pheromone receptors belong to this class

• A prototypical eukaryotic signalling system is the MAP (mitogen activated protein)

kinase cascades;

• S. cerevisiae has at least six such pathways, which together control cellular

morphology and responses to pheromone and environmental stress

• limitations for instance S. cerevisiae is lacking receptor tyrosine kinases or nuclear

receptors, important classes of mammalian hormone receptors

Model character: signal

transduction

Model character: control of

gene expression• The principles of the

control of transcription are well conserved across eukaryotes

• The organisation of the transcription initiation machinery seems to be conserved, i.e. there are counterparts for most if not all subunits in yeast and human

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

Model character: the

unexpected• Prions

– Have of course been in the focus of interest through mad cow disease

– Yeast also has two systems that seem to have all features of prions!

– Ageing

– Yeast cells have a pre-determined life span, i.e. mother cells die after a

certain number of divisions

– There is also a ”common” gene, WRN (Werner’s syndrom) in human

and SGS1 in yeast; the genes are homologous and mutations causes

premature ageing in human and yeast, respectively

Yeast biotechnology:

heterologous expression• The production of proteins is of interest for several purposes:

– For research, such as for purification and structural analysis

– For industry, such as for the production of enzymes for the food and paper industry or for research and diagnostics

– For the 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 (or may not) perform the same or at least similar post-translation modifications, such as glycosylation

• Yeast usually reaches only a lower level of expression: up to more than 50% of the cellular protein have been obtained in E. coli systems but no more than 10-20% even in the very best yeast system

• 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

Heterologous expression in

yeast: drug screening• Replace pheromone receptor with human

receptor

• Allow human receptor to drive signal cascade

pathway to activate a reporter gene.

• Can identify drugs that interact with the human

receptor.

• Can identify antagonists that prevent drug

binding

READING MATERIAL

Getting Started with Yeast

By Fred Sherman

Modified from: F. Sherman,

Getting started with yeast,

Methods Enzymol. 350, 3-41

(2002).