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M.W.W., D. Stone, and E. Craig

Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae. M.W.W., D. Stone, and E. Craig. W hat’s the question?. Do subfamilies (based on sequence identity/homology) of multi-gene families have separable functions?

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M.W.W., D. Stone, and E. Craig

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  1. Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae M.W.W., D. Stone, and E. Craig

  2. What’s 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?

  3. 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.

  4. 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 didn’t 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.

  5. 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.

  6. What was known? • SSA1 and SSA2 (96% identical) could be knocked out with no change in phenotype. If both of them were KO’d, 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)

  7. What’s 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 can’t 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.

  8. 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?

  9. 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?

  10. When are SSA3 and SSA4 expressed?Northern blot

  11. Construction of ssa3 and ssa4 disruptions

  12. Strain Construction

  13. Were the genes disrupted?Southern blot

  14. Could we identify the gene products?

  15. 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?

  16. Were all spore types viable?

  17. Can we determine that the “missing” spore is ssa1ssa2ssa4?Southern blot

  18. Plasmid constructs for rescue

  19. Is it possible to artificially construct a conditional mutation? Transform into heterozygous quadruple mutants a plasmid containing a GAL1p:SSA1 construct. Galactose is the permissive condition; Glucose is the non- permissive condition

  20. 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.

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