Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevis

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Complex interactions among members of an
essential subfamily of hsp70 genes in
Saccharomyces cerevisiae

• M.W.W., D. Stone, and E. Craig

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Whats the question?

• Do subfamilies (based on sequence
  identity/homology) of multi-gene families have
  separable functions?
• Are heat-shock genes essential under
  non-heat-shock conditions?

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NB The role of an introduction

• Please note when you are writing your paper this
  is the job of any introduction of a scientific
  paper to give background and rationale for why
  the experiments described in the paper were done.
• It is also the job of the introduction to get the
  reader interested in why the work was done.

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What was known?

• Had found two genes in Drosophila and assumed
  yeast would be simpler.
• Found 8 related genes in yeast
• Most people assumed HS genes were essential for
  HS but probably didnt have a major role during
  normal growth.
• Based on homology, we could see there were likely
  to be a few subfamilies, but there was no idea
  what these genes might be doing.

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Heat shock

• It had been found that when cells of a number of
  organisms were given a prior incubation at a high
  but sublethal temperature prior to incubation at
  a normally lethal temperature, they could
  survive.
• In studies of Drosophila, it had been noticed
  that new puffs appeared in chromosomes during a
  heat shock.
• By isolating RNA from flies after a heat shock
  and identifying segments of DNA that encoded
  these genes, the first heat-shock genes were
  cloned.
• Because this was years before sequencing of
  entire organisms, the number of HSP70-related
  genes had to be deduced by low-stringency
  northern blots.

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What was known?

• SSA1 and SSA2 (96 identical) could be knocked
  out with no change in phenotype. If both of them
  were KOd, cells formed small colonies, were
  temperature sensitive and, paradoxically,
  resistant to heat shock.
• Two-dimensional protein gels showed that many
  heat-shock proteins were constitutively induced
  in an ssa1ssa2 double mutant.
• SSA3 and SSA4 were the next most closely related
  genes (80 identity)

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Whats the approach?

• Molecular biology
• You needed to knock out one gene at a time, then
  construct multiple mutants by mating.
• Because it is impossible to study mutants that
  are dead, i.e. that you cant grow, we needed to
  construct a strain that could be grown and then
  lose the essential gene.
• At the time, yeast was the only organism in which
  this was possible and this was the first time
  multigene families had been studied in this
  fashion.

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Approach

• A few years earlier, Rodney Rothstein from
  Columbia University had developed a technique for
  knocking out genes by homologous recombination.
• Again, no PCR, so we had to depend primarily on
  restriction sites that were present in the gene.
• Homework (look up restriction sites at SGD for
  SSA1)
• Had to use the untranslated regions of these
  genes for knockouts why?

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Methods

• If SSA1 is only 2.5 kb, why was a 6.6 kb,
  PvuII-BamHI used for disruption?
• Already had ssa1ssa2 double mutant
• Needed to construct ssa3 and ssa4 mutants and
  cross them all to get multiple mutant.
• Needed to have different selectable markers for
  each gene if possible. Why?

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When are SSA3 and SSA4 expressed?Northern blot
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Construction of ssa3 and ssa4 disruptions
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Strain Construction
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Were the genes disrupted?Southern blot
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Could we identify the gene products?
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Mating to obtain triple and quadruple mutants

• After sporulation, expect ½ of the spores to
  contain a mutation
• With two genes, the chance of finding two alleles
  of a specific genotype, e.g. a double mutant is ¼
  or ½ X ½
• The chances of finding three mutant genes
  together is ½ x ½ x ½ or 1/8
• The chance of finding four mutant genes is 1/16.
• There are four spores in a tetrad, so how often
  in 4 tetrads would you expect to find a triple
  mutant or a quadruple mutant?

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Were all spore types viable?
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Can we determine that the missing spore is
ssa1ssa2ssa4?Southern blot
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Plasmid constructs for rescue
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Is it possible to artificially construct a
conditional mutation?
Transform into heterozygous quadruple mutants a
plasmid containing a GAL1pSSA1 construct.
Galactose is the permissive condition Glucose is
the non- permissive condition
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Conclusions

• ssa3 and ssa4 and ssa3ssa4 mutants exhibited wild
  type phenotypes.
• SSA1,2,3, and 4 make up a phenotypically
  identifiable subfamily
• There were three, phenotypically identifiable
  subfamilies SSA, SSB, and SSC.