Yeast as a Model System

MBIOS 520/420

September 29, 2005

Advantages of Yeast as a Model

• Geneticists are always interested in humans, but it is impractical (and unethical) to do human studies

• Even mouse models & cell cultures can only take us so far:

Yeast Mammalian Cell Culture

• 2 hour generation time

• Can be maintained as either haploid or diploid

• Small genome

• 24 hour generation time (after 4 week culturing)

• Always diploid

• Very large genomes

Despite the differences, yeast cells are organized in a very similar manner to higher eukaryotes

(organelles, chromosomes & proteins are similar)

The Yeast Genome

THE MATH

Yeast Human

14,000,000 bp

933 clones (1X λ library)

5600 clones (6X λ library)

70 clones (1X BAC library)

933 clones (6X BAC library)

3,500,000,000 bp

233,333 clones (1X λ library)

1,400,000 clones (6X λ library)

17,500 clones (1X BAC library)

105,000 clones (6X BAC library)

Identification of Yeast Biosynthetic Genes

• Cloning of biosynthetic genes aided both the study of cellular metabolism, and provided phenotypic genetic markers

• Biosynthetic genes = those involved in production of cellular metabolites like amino acids

• Because these are basic needs of all cells, the yeast genes are similar enough to the E. coli biosynthesis genes

• Researchers introduce random pieces of genomic DNA into E. coli mutants, until function is restored

• This is termed genetic complementation

Genetic Complementation – Cloning the LEU2 Gene

Isolate yeast genomic DNA.

Cut up the genome and ligate strands into a plasmid.

Transform large numbers of E. coli with the entire library.

Grow the cells on media lacking leucine.

Only colonies with functional leucine-producing yeast

genes will grow.

Identification of Yeast Biosynthetic Genes

• Once identified, a biosynthetic gene like LEU2 can be used to

complement yeast cells that are leu- as well as E. coli

• A plasmid carrying LEU2 could then be used with leu- strains in the same way that ampicillin resistance is used as a selectable marker for E. coli

• But how do we identify biosynthetic mutants in the first place?

• Can we use selective media? You can’t isolate a colony that doesn’t grow.

• The answer replica plating

Replica Plating

Plate colonies on normal media with leucine.

Place a piece of velvet (or silk) over the plate & the colonies

will transfer.

Press this “carbon copy” onto a new plate w/o leucine.

See which colony doesn’t grow & pick it off the old plate.

With Leu w/o Leu

Leu-

mutant

replica

Yeast Vectors

• Three major types (each useful for a different application)

• Simplest

• LEU2 or similar marker

• Has bacterial ori & ampr

(derived from pUC vector)

• Cannot replicate in yeast

• Limited to single copy(sometimes useful)

• Gene must integrate into yeast genome to be stably expressed

Integrating Plasmids

Yeast Vectors

• Shuttle vectors(replicate in yeast & E. coli, has ampr & LEU2)

• One of two yeast ori sequences can be used

• 2μ = yeast plasmid

• ARS = autonomously replicating sequence (yeast chromosomal DNA) (less stable)

• CEN = centromere DNA, makes the yeast cell treat the plasmid like a chromosome at mitosis (1 copy only)

Replicating Plasmids

Yeast Vectors

• Linear vector

• Has yeast telomere sequences at each end

• Replicates and is inherited like a chromosome

• Capable of large inserts(300 kb)

• Similar to BAC (just a very large plasmid)

Yeast Artificial Chromosomes

Cloning Genes by Genetic Complementation• Useful for identifying conditional lethal mutations

• Biosynthesis or temperature sensitive mutations for ex.

After isolating mutants, transform them with the entire

genomic library.

Plate them w/o leucine to detect transformants.

Replica plate them and grow under conditions to ID mutant

(ex: higher temperature).

Isolate & sequence the colonies that survive.

Sub-Cloning• When we isolate a BAC or plasmid with a gene of interest, we must do sequencing to find the gene within the vector

• Modern sequencing only allows us to sequence 0.5 kb at a time(sequencing is like PCR, proceeds from a known primer)

• We need to sub-clone large inserts like BAC/YAC (~200 kb) or a lambda insert (~15 kb) by transferring them to plasmids

• The only other way would be to primer walk for our sequencing(very time consuming)

• We can either sequence all these, or do another genetic complementation on the sub-cloned sequences

Sub-Cloning To Find A Gene

We’ve found a YAC with Gene X from our genetic complementation screen.

Cut the YAC insert with restriction enyzmes.

Put these smaller fragments into plasmids (< 4 kb)

We would want to cut a 200 kb YAC fragment into 50 pieces to

sequence get 4 kb inserts. Choose an appropriate enzyme.

Introduce back into yeast & repeat complementation

test.Sequence the survivor.

Potential Problem?

RestrictionDigest

Ligate into plasmids

TransformGrow on leu-,

high temp

Sequencethis one!

Gene X

Yeast-Two-Hybrid Assay - Basis

• Used to detect genes that encode proteins that interact with each other (one is “bait” the other is “target”)

• Ex: Let’s say we’ve cloned Gene X for the first time. We know it binds to DNA, and we suspect it binds to another protein, but which one?

• We are using a transcription factor as an example, but this assay will work with any proteins that bind to each other

• The basis of this technique is the two domains of transcription factors the DNA binding domain and the activation domain

• In domain swapping experiments, we saw that we could fuse the DNA binding domain of one TF with the activation domain of another

Yeast-Two Hybrid Assay - Background

GAL4 promoter is fused with lacZ reporter gene.

The binding domain (BD) & activation domain (AD) act

together to start transcription.

We can place this in a vector and create blue colonies when X-Gal is present.

GAL4DNA Binding

Domain

GAL4Activation Domain

RNA Polymerase

LacZ GenePromoter

Promoter

MCS of plasmid vector

GAL4 BD GAL4 AD

Yeast-Two Hybrid Assay - Procedure

When we transform yeast cells with this, RNA

polymerase is not activated. Protein X can’t bind to RNA polymerase. Transcription

not activated.GAL4

DNA Binding Domain

Protein X

LacZ GenePromoter

Promoter GAL4 BD Gene XFirst we replace the GAL4 AD with Gene X.

Colonies are white.

Yeast-Two Hybrid Assay - Procedure

Next we fuse Gene Y to GAL4 AD.

Then we transform yeast cells with two vectors:

1)Carrying GAL4BD-GeneX fusion

2)Carrying GeneY-GAL4AD fusion

Let’s say Gene Y binds to Gene X.

Protein X & Y bind to each other, bringing

GAL4 AD close to RNA Polymerase.

Promoter GAL4 BD Gene X

Vector 1

Promoter Gene Y GAL4 AD

Vector 2

GAL4BD

Protein X

LacZ GenePromoter

Protein Y GAL4 AD

Transcription occurs. Colonies turn blue.

Yeast-Two Hybrid Assay

Summary

Gene X and Gene Y bind to each other.

Fuse Gene X to GAL4 BD & Gene Y to GAL4 AD.

Transform yeast with two vectors, each carrying one of these fusion proteins.

The GAL4 BD binds to DNA, along with the fused Protein X.

Since Protein Y binds well to Protein X, it will bind near the promoter as well.

Since GAL4 AD is fused with protein Y, it is brought close enough to the promoter to activate RNA polymerase &

transcribe lacZ. This proves that Proteins X & Y bind together.

Yeast-Two Hybrid AssayYeast-two hybrid is effective at demonstrating that two

proteins interact, but what if we have no idea what protein Gene X binds to? How do we find that gene?

STEP1. Make a cDNA library of your

organism.

STEP2. Fuse every cDNA in your library with the GAL4 AD.

Isolate mRNA

Reversetranscribe

Put in vectors

(thousands of cDNAs)

NOTE: All vectors should have LEU2 selectable marker.

Yeast-Two Hybrid Assay

STEP3. Transform yeast cells with the

GAL4BD-GeneX fusion gene.

NOTE: This vector should have a selectable marker

different from LEU2!

w/o tryptophan

Transform

TRP1

STEP4. Transform yeast cells again, this time with the GAL4AD-cDNA fusion

genes.

STEP5. One of the cDNAs fused with GAL4 AD will

bind with Gene X. This one will turn activate

transcription & create a blue colony.

w/o trp or leu

Isolate this

colony. Sequence

cDNA.

(thousands of cDNA-GAL4 combinations)

Transform

LEU2