Molecular Biology

•  Yeast Transformation

•  Yeast Plasmids

•  Gene Disruption, tagging

•  Cloning by Complementation

•  Epistasis

Transformation •  Transformation: introduction of DNA 1978, ca 1000x less efficient than E. coli

–  Selectable marker

–  Spheroplasts, Li2+ salts, electroporation

•  Yeast plasmids are shuttle plasmids, i.e. can be propagated in yeast and E. coli,

–  E. coli: ori, β-lactamase

–  Yeast: CEN, URA3

Different properties for different uses

Yeast Plasmids

4 types of yeast vectors

Homologous recombination is very efficient in yeast. Is the knock-out strain viable? What is the phenotype of a strain without the gene of interest?

GENE

SELECTABLE MARKER

SELECTABLE MARKER

Gene-disruption

The Saccharomyces Genome Project has revealed the presence of more than 6000 open reading frames (ORFs) in the S. cerevisiae genome. Construct a set of deletion mutants corresponding to all these ORFs. The method used was a PCR-based gene deletion strategy to generate a start- to stop- codon deletion of each of the ORFs in the yeast genome. Each gene disruption was replaced with a KanMX module and uniquely tagged with one or two 20mer sequence(s) . The presence of the tags can be detected via hybridization to a high-density oligonucleotide array, enabling growth phenotypes of individual strains to be analyzed in parallel .

Adding a Bar Code

Heterozygotic Disruption Collection

•  Deletion mutants available in haploid or diploid backgrounds (each ~4’500 strains).

•  This work uses heterozygous knockout collection.

•  Haploid or homozygous knockout typically has clearest phenotype.

•  Why is this library used ?

Cell Volume 116, January 2004, Pages 121-137

Gene-tagging •  In a similar way, a gene can be tagged. For

instance, if the cassette is inserted in frame to the end of the ORF it will generate a fusion protein, with lacZ, GFP or an immuno-tag for protein detection and purification

•  For instance, there are now sets of strains available in which each yeast has been tagged with GFP or TAP-tag

YFG1 URA3 GFP

Localising proteins in the cell: GFP •  The green-fluorescent protein is used now systematically to localise

proteins within the yeast cells •  A main advantage of the GFP technology is that it allows watching

processes in the living cell ! •  Usually the coding sequence of GFP is fused to the end of the coding

region of the gene of interest •  This can be done on a plasmid but also within the genome •  The resulting construct is tested for functionality by complementing the

corresponding deletion mutant •  GFP shines green in the fluorescence microscope and the subcellular

localisation can be deduced using control staining of different compartments

•  There are now many different versions of GFP with different detection threshold and different emission colours: CFP, RFP, YFP...

•  This allows simultaneous observation of several proteins in the cell and even protein-protein interaction

yeastgfp.ucsf.eduYEAST GFP FUSION LOCALIZATION DATABASE

>> Advanced Query < Go >

>> Quick Search yal001c

clr

Welcome to yeastgfp.ucsf.edu

The database of our global analysis of protein localization

studies in the budding yeast, S. cerevisiae.

> quick case-insensitive searches of the database may be performed on yeast orf

names (yal001c) or gene names (TFC3)

> separate multiple orfs/genes with a space (e.g. yal001c zwf1 bud2 etc.)

> more advanced searching and downloading can be done in Advanced Query

> GFP-tagged strains can be obtained from Invitrogen.

> TAP-tagged strains can be obtained from Open Biosystems.

> more details available in >> info >> faq >> help

This web site supports Huh, et al., Nature 425, 686-691 (2003). <pdf>

The quantitation data presented here is published in Ghaemmaghami, et al., Nature 425, 737-741 (2003). <pdf>

Detailed collection construction methods can be found in Howson et al., Comp Funct Genom 6, 2-16 (2005). <pdf>

This research is the work of the laboratories of Erin O'Shea and Jonathan Weissman at the University of California San Francisco.

Please direct comments, concerns, and questions to <jan.ihmels@gmail.com>

© Copyright 2001 - 2006 University of California Regents. All rights reserved.

•  50% of all genes have NO DETECTABLE

PHENOTYPE when disrupted. •  Not all important pathways are essential. •  Redundant genes may exist. •  The robustness of yeast is only partly due

to redundant genes. •  The problem in figuring out the function of

a non-essential gene is determining what question to ask!

Only a very small percentage of genes are essential (1000 of 6000)

How to deal with essential genes ?

•  GAL-YFG1, Tet-YFG1

•  Ts alleles

•  Degron

•  DAmP (decreased expression of essential genes through mRNA perturbation)

•  DAmP + Degron

Integration of plasmids into the yeast genome

•  Integration occurs by homologous recombination, this means that a plasmid like YIp5 will integrate into the URA3 locus

•  Integration results in the duplication of the target sequence •  The duplicated DNA flanks the vector •  Integration can be targetted by linearisation within one of the sequences: cut

DNA is highly recombinogenic •  Integrated plasmids are stably propagated but occasional pop-out by

recombination between the duplicated sequences

URA3 ura3 X

URA3

ura3 X

plasmid

genome

genome

Marker rescue •  By counterselection agains URA3 on 5-flouro-orotic acid,

which is toxic to URA3 cells •  Or use of a Cre/loxP recombination system •  Very important for multiple deletions, i.e. 7-fold knockout

strains

URA3

URA3 plasmid

genome YFG1

YFG1

•  Omportant if one wants to re-use the marker in order to make many deletions in one and the same strain (there are strains with more than 20 deletions!)

•  It is also important for industrial yeast strains; when one wants to engineer those at the end no foreign DNA should be left behind (but for hardliners on genetic engineering the intermediate presence of foreign DNA ina yeast is already ”dangerous”)

•  All these methods use homologous recombination a second time, i.e. to pop-out the integrated DNA again

•  An example for this are the loxP-kanR-loxP cassettes; recombination between the two loxP cassettes is stimulated by the Cre-recombinase (transformed on a separate plasmid); recombination just leaves behind a single loxP site

•  Also used for conditional, time- or tissue specific knockouts in mice

The Cre/loxP system

Cloning in yeast by gap repair

X

Xgapped plasmid

Gene fragment

X Xgenomic copy is used to repair the gap;

the template is duplicated. Allele rescue

YFG1

YFG1

Plasmid shuffling

Cloning by complementation

•  How do you clone the gene that you have mutated and is now present in your mutant ?

•  You clone it by complementing the mutant phenotype by transformation with a genomic library.

Cloning by Complementation:

Transform yeast mutant with library of expression plasmids and screen for return to wild type phenotype

Recover plasmid, shuttle into E. coli, sequence the clone

Celebrate?!

Must prove is ‘the gene’ and not a suppressor

You’ve cloned the gene. Now what?

Define the pathway by defining interacting genes i.e., suppressors and enhancers of original mutation

Types of suppressors:

Allele specific --will only suppress specific point mutations may identify physical interactions

Bypass-- will suppress loss of gene

may identify downstream factors High copy-- overproduction of one gene product

compensates for loss or defects in another often identifies upstream factors

Pbs2p and Hog1p are in the same pathway and Hog1p is activated by Pbs2p. Overexpressed Hog1p may confer sufficient activity to mediate the required function even in the absence of Pbs2p.

X

common target Two parallel pathways share one or several common targets. Overexpression and hence higher activity of the parallel pathway may be sufficient to activate the target.

X

Getting further: synthetic lethality

•  Synthetic lethality is a powerful method to identify genes whose products operate (in a pathway) parallel to the one that is affected by the primary mutation

•  Typically, the primary mutant is transformed with a plasmid that carries the corresponding gene; the gene is either expressed through the GAL1 promoter (i.e. ”on” on galactose and ”off” on glucose) or is on a plasmid with URA3 as marker, which can be counter selected with 5-FOA

•  Mutations are then screened that cause the yeast to grow only in the presence of the plasmid (i.e. not on 5-FOA) or only when the gene is expressed (i.e. not on glucose)

•  The principle approach is so powerful that synthetic lethality screens are now done at a genome wide scale using the yeast deletion mutant collection: this means 4,200 x 4,200 crosses, sporulations and tetrad analyses done by robotics

Synthetic lethality

X

X

common target

X

common target

The two pathways control some common targets; mutation of PBS2 alone causes only a moderate phenotype. The second mutation in the parallel pathway leads to lethality -> see SGA Analysis

Getting further: epistasis I •  Establishing the order of genes in a pathway •  Generate a double mutant; the phenotype of the double mutant may

reveal if the two gene products work in the same or in parallel pathways and they may reveal the order within a pathway.

X

X

common target

•  mutation in all these four proteins cause similar phenotypes, such as moderate sensitivity to salt

•  When we combine the hog1 and the pbs2 in a hog1 pbs2 double mutant then we would expect that the double mutant has the same level of sensitivity as each single mutant; we would conclude that they function in the same pathway

•  When we combine the pbs2 and the cba1 mutation in a pbs2 cba1 double mutant we would expect a strongly enhanced sensitivity of the double mutant as compared to the single mutants; we would conclude that Hog1p and Cba1p work in different, though parallel pathways

Getting further: epistasis II •  Let us now assume that deletion of PBS2 (and of HOG1)

causes sensitivity to high salt concentrations while deletion of SKO1 causes higher tolerance to salt in the medium

•  If those proteins act in the same pathway there are different possibilities for the phenotype of the pbs2 sko1 or hog1 sko1 double mutant

•  If Sko1p were downstream of Pbs2p and Hog1p we would expect that the double mutant is tolerant, i.e. has the same phenotype as the sko1 single mutant: sko1 would be epistatic (”dominant over”) to pbs2 and hog1 (and this is really the case)

•  If Sko1p were upstream of Hog1p and Pbs2p we would expect that the double mutant pbs2 sko1 and hog1 sko1 is sensitive to salt

X

X Sko1

Getting further: epistasis III • Also multi copy suppression or activating mutations are

useful tools in epistasis analysis • Suppression by overexpression can only work for a gene/

protein functioning downstream of the primary lesion, as indicated here for Hog1p; overexpression of PBS2 would not suppress a hog1∆ mutation

• In a similar way, an activating mutation of HOG1 can suppress the salt sensitivity of a pbs2∆ mutant, but not vice versa, and this is indeed exactly how it works

• The epistasis concept has been used in very many examples to analyse the order of events in signalling pathways and other cellular systems: if the phenotype of the double mutant resembles that of one of the single mutants the latter gene product functions further downstream in the system, i.e. closer to the physiological effect

X

X

Getting further: suppressor mutations •  An extragenic suppressor mutation alters a different gene product

such that the, or one of the, effects of a certain mutation are overcome

•  Like with multi-copy suppression there are many ways in which this can happen and the outcome of such an approach is often quite surprising but very informative

•  Typical suppressor mutations are those that activate a gene product downstream of the primary lesion in the same pathway; since such mutations cause a gain of function they are usually dominant

•  Other typical suppressor mutations knock out a repressor downstream in the same or in a parallel pathway; since such mutations cause a loss of function they are recessive

•  A suppressor mutation may also activate or inactivate pathways/systems that affect in some way the same physiological system than the primary lesion

•  If a given protein is part of a multimeric complex and the primary mutation is a point mutation, extragenic suppressor mutations might occur such that protein interactions are restored; hence this is a method to identify interacting proteins

Getting further: suppressor mutations

X

Pbs2p and Hog1p are in the same pathway and Hog1p is activated by Pbs2p. A mutation that renders Hog1p active even without activation would suppress the pbs2 mutation and is probably dominant

The pathway ultimately inactivates a negative regulator, e.g. the repressor Sko1p; knock out of the repressor could overcome inactivation of the pathway; the mutation is most likely recessive

X

X Sko1

Interplay between genetics and biochemistry/molecular biology

•  Classical genetics Mutant -> gene -> molecular function

-> new mutants in given pathway -> new gene etc. until pathway is “resolved”

•  Reverse genetics: Gene, i.e., from genome sequencing project -> phenotype -> function (annotation)

Mutagenesis in vitro

•  Structure <-> function study of a protein by site directed mutagenesis

•  Generation of ts allele by error prone PCR

•  Plasmid shuffling