| Summary: | The aim of this project is to identify and characterise novel client proteins of Cdc37 in <i>S. pombe</i> through a synthetic lethal genetic screen. During the screen, 68 strains were initially picked out of 40,000 colonies as potentially synthetically lethal with <i>cdc37-681</i>, a temperature sensitive allele, at permissive temperature. When retested, 12 strains were identified as candidate strains. Mutations identified in 3 strains were found by genetic analysis to have a phenotype of their own in a <i>cdc37<sup>+</sup></i> background. A genomic library was transformed into each synthetic lethal mutant. <i>wis4, msc1, nak1 </i>and <i>cdc7</i> were identified as candidate genes that rescue the various mutants. <i>wis4</i> encodes a MAP3K involved in the stress-responsive signal transduction pathway; <i>win1</i> encodes a closely related kinase. By crossing <i>wis4</i> and <i>win1</i> deletion strains with <i>cdc37-681,</i> it was proved that <i>wis4</i> and <i>win1 </i>are not synthetic lethal with <i>cdc37-681. msc1</i> is a multi-copy suppressor of <i>chk1</i> and has a role in regulating chromatin structure. However, neither <i>musc1</i> nor <i>chk1</i> is synthetically lethal with <i>cdc37-681</i>. <i>nak1</i> encodes an essential protein kinase and plays a role in the regulation of cell polarity, growth and division. But <i>nak1ts</i> mutants do not show synthetic lethality with <i>cdc37-681</i>. Cdc7 is a protein kinase essential for septation and cell division. A known <i>cdc7ts</i> mutant, <i>cdc7-24</i> was shown to by synthetically lethal with <i>cdc17-68.</i> The synthetic lethal mutation outcrossed from J322 (the strain rescued by <i>cdc7<sup>+</sup>)</i> shows the same phenotype as <i>cdc7-24</i>. Cdc7 is a possible Cdc37 client, but Cdc7 protein levels do not change in <i>cdc37-681 </i>or <i>cdc37-184</i> at restrictive temperature. Cdc7 kinase activity is produced in <i>cdc37-681</i> and <i>cdc37-184</i>, indicating that Cdc37 function is needed for Cdc7 kinase activity. The cell morphology of <i>cdc37ts</i> in combination with GFP-tagged <i>cdc7</i> is similar to that of <i>cdc7ts</i> mutants and differs from the <i>cdc37ts</i> single mutant. Cdc7 can locate to the spindle pole body in <i>cdc37ts</i> strains, which suggests Cdc7 localisation does not require Cdc37.
|
|---|
