Answer Key for Exam on Yeast Genetics and Protein Expression. Dr. Nelson April 10, 1997

Since there is a lot of material covered in this exam, and there have been three lecturers that 
will ask questions, please keep your answers short.  You should not need to spend more 
than 5-10 minutes on any question.  (5 points per question, 60 points total)

1) What is the difference between a two hybrid screen and a one hybrid screen?

A two hybrid screen uses protein fusions between separate DNA binding domains and 
activation domains to search for protein-protein interactions that will turn on a reporter 
gene.  A one hybrid screen uses only fusions to an activation domain and a known DNA 
sequence placed upstream of the reporter gene.  This system looks for protein-DNA 
interactions with the known DNA sequence.

2) What are two potential problems that can arise later if you do a gene disruption instead of   
     a gene deletion?

The disrupted gene on the chromosome can recombine with a mutant on a plasmid to 
restore a wild type sequence either to the chromosome or to the plasmid.  

The N-terminal part of the disrupted gene may still have some activity leading to false 
conclusions about the phenotype of a null mutation.

3) Why do some high copy suppressors of a temperature sensitive mutant work, when a 
     low copy vector carrying the same gene cannot?

A high copy suppressor often means there is a direct protein-protein interaction between the 
ts mutant and the suppressor gene product.  This interaction is weakened by the ts mutant 
and it can be stabilized by higher concentrations of the suppressor.  If the suppressor is a 
redundant gene that does the same thing as the mutant, then you should not have seen the ts 
phenotype in the first place, unless the expression level or the activity of the suppressor is 
much lower than the normal activity of the mutated gene.  In this case higher concentrations 
of the protein may overcome lower activity levels.  

Eamples:  AAC1 is an ADP/ATP carrier of yeast.  It translocates ADP and ATP across the 
inner mitochondrial membrane.  Normally it is made at low levels and it cannot replace 
AAC2 (the main ADP/ATP carrier) in single copy.  It can complement an AAC2 knockout
on a high copy plasmid.  These proteins do not interact.

TOM6 is a protein component of the mitochondrial import apparatus for protein import.  It 
was cloned as a high copy suppressor of a TOM40 ts mutant.  TOM40 is another 
component of the same import apparatus.  These proteins interact in the complex.  

4) What is a synthetic lethal phenotype and why would you ever do a synthetic lethal 
     screen?

Synthetic lethal means that a redundant gene that has no phenotype when it is deleted is 
made essential by mutation of its redundant partner.  This is followed in yeast by the ability 
or non-ability of yeast to lose a plasmid carrying the gene in question.  An ADE1 or ADE2 
selectable marker is present on the plasmid to produce a color change from white to red 
when it is lost.  Yeast that have been mutated so a plasmid can no longer be lost (producing 
red sectors in a colony) can be used to clone the redundant gene by complementation.  This 
restores sectoring, as the synthetic lethal gene can now be lost again.

A synthetic lethal screen is used to identify redundant genes.

5) All the genes on yeast chromosome VIII are being replaced from start codon to stop 
    codon with green fluorescent protein.  What can be learned from these GFP knockouts?  
    What could be learned if the GFP was fused at the C-terminal of the protein rather than 
     replacing it?

The GFP protein will be made from the deleted gene's promoter, so the expression of the 
deleted gene can be tested under a variety of growth conditions by simply looking for the 
fluorescence under the microscope.  Genes that are only expressed during meiosis, 
sporulation, respiration, mating, heat shock, etc. will only fluoresce under those 
conditions.

If the protein is essential, that will be obvious from tetrad dissection starting with a 
heterozygous diploid strain.  There will be a 2:2 survival pattern in the four spores.  

Fusion at the C-terminal will allow the fluorescence signal to be targeted where the native 
protein would normally go.  A nuclear protein would go to the nucleus and show up as 
fluorescent nuclei in the microscope.  This won't work with proteins whose targeting 
signal is on the C-terminal (like peroxisomal proteins SKL or ER lumenal proteins KDEL).

6) Shuttle mutagenesis, as practiced by Mike Snyder's lab and by Kristin Chun and Mark 
     Goebl, uses a transposon to mutagenize yeast genes.  The genetic footprinting method 
     of Smith, Botstein and Brown also uses a transposon.  Each method has advantages and 
     disadvantages.  Give one advantage of each method as contrasted with the other 
     method.

The shuttle mutagenesis method results in individual strains that can be identified by 
recovery of the transposon containing an amp resistance marker and sequencing of the 
yeast DNA.  This allows construction of a set of individual knockout strains.  The genetic 
footprinting method is a batch process and does not result in isolated knockouts.

The genetic footprinting method allows for the monitoring of expression of any yeast gene 
under any growth condition without making a specific knockout of that gene.  It is a highly 
efficient way to evaluate phenotypes, even if they only confer a 5-10% reduction in fitness.  
The shuttle mutagenesis system is dependent on individual disruptions for analysis so it is 
more labor intensive to get similar results.

7) Unfortunately, many eukaryotic proteins are toxic when expressed in E. coli.  How did 
    William Studier and colleagues use T7 lysozyme to reduce expression of toxic genes in
    E. coli until they were induced?

The pET vectors have T7 RNA polymerase under control of the lac UV5 promoter that is 
inducible by IPTG.  However, this system is a bit leaky.  To reduce the leakyness, Studier 
made a second plasmid that coded for T7 lysozyme, an inhibitor of T7 RNA polymerase.  
Expression of this lysozyme (high amounts pLysE, or low amounts pLysS) reduced the 
leakyness of the system by inhibiting T7 RNA polymerase that is made before induction. 

8) Give two reasons why purification of an expressed protein by the intein based IMPACT 
    system might not be a suitable method for some proteins.

Unstable to overnight elution conditions.  
C-terminal residue is critical for function.
Protein requires mammalian or eukaryotic post translational modifications.
Many disulfide bonds in the protein that will be broken by DTT elution.

9) How is LacZ used as a reporter in the baculovirus system?

LacZ is present on the transfer vector and the linearized AcMNPV viral genome.  The only 
way to get active beta galactosidase is by recombination of the trasfer vector and the 
AcMNPV DNA.  Such a system will not make occlusion bodies (occ-) but it will be blue 
on XGAL plates.  This provides a simple screen for recombinant virus plaques.

10) If you want to overexpress an eukaryotic membrane protein, which system would you 
      choose and why?   S. cerevisiae, baculovirus, Pichia pastoris, ecdysone inducible 
      mammalian system.

My preferred system would be Pichia since the peroxisomal membrane is amplifed greatly 
in the methanol induced cells.  This is an ideal target for a membrane protein if the normal 
targeting signal can be overidden by adding a peroxisomal targeting signal to the C-terminal 
of the protein.  

Baculovirus might also be a good system since it has a built in capacity for overproduction.  
The mammalian inducible system is probably not a good choice for making a lot of protein, 
because of the cost of cell culture, however, posttranslational modifications may be 
essential to function and these might not be achieved correctly in the other systems.  Yeast 
does not overproduce to a great degree, and it has lots of problems with proteases.  You 
might want yeast in spite of these problems if you are working on a yeast protein, or if you 
would benefit from the yeast genetic tools available.

The key to this question is your justification.  If you make a good argument for your 
choice, you could receive full credit for any of these systems.

11) If you wanted to study the effects of turning a yeast gene on or off what promoter 
       would you choose and why?

My choice would be the steroid inducible expression system, but the gal 1, 10 promoter is 
probably the most commonly used method.  Both methods allow control of expression by 
induction either with galactose or steroid.  Glucose represses the Gal 1,10 promoter so it 
actively shuts down expression.  Removal of steroid from the media will have the same 
effect.  The Gal 1,10 system is a bit leaky, so the steroid system may provide cleaner 
results, especvially if youe protein is only required in small amounts.

12) What makes methylotrophic yeast like Hansenula polymorpha and Pichia pastoris such 
       potent expression systems?

The AOX1 promoter is such a strong promoter that a protein made from this promoter and 
induced by methanol can make up to 30% of the cell's protein.