Micro-controladores

  There   have   been   a   number   of   genetic   screens   performed   that   have   identified  genetic  interactions  of  HSP12  and  HSP26    (Addinall   et   al.,   2011,   Duennwald  et  al.,  2012,  Moehle  et  al.,  2012,  Costanzo  et  al.,  2010,  Sharifpoor  et  al.,   2012,   Krause   et   al.,   2012).   In   these   genetic   screens  HSP12  and  HSP26  have   been   identified   as   the   prey   from   other   gene   baits,   so   only   a   fraction   of   the   potential   genetic   interactions   have   been   tested.   In   contrast,   this   is   the   first   study   that   has   identified  the  genetic  interactions  of  HSP12  and  HSP26  when  these  genes  are  used   as   the   genetic   bait.   This   study   has   only   focused   on   identifying   the   genetic   interactions  of  HSP12  and  HSP26.  This  differs  to  other  genetic  screens  which  have   analysed   up   to   5.4   million   gene-­‐gene   interactions   providing   genetic   interaction   profiles  for  approximately  75%  of  all  genes  in  S.  cerevisiae  (Costanzo  et  al.,  2010).   Since  this  study  was  focused  on  only  two  genes  the  genetic  interactions  identified   are   more   likely   to   be   reliable   than   those   generated   from   genome-­‐scale   screens   which  may  be  prone  to  more  mistakes.    

  In   this   study   only   negative   genetic   interactions   were   considered.   Negative   genetic   interactions   were   easier   to   visualise   from   the   colonyzer   images   than   positive  genetic  interactions.  Also  strains  growing  nearby  to  strains  with  a  synthetic   sick   or   lethal   genetic   interaction   grew   better   because   of   an   increase   in   the   availability  of  nutrients.  These  strains  may  therefore  look  to  have  a  positive  genetic   interaction   and   give   a   positive   GIS   when   this   increase   in   growth   is   actually   not   a   result   of   a   positive   interaction   between   two   genes.   In   addition,   negative   genetic   interactions   were   easier   to   re-­‐confirm   when   performing   spot   tests.   Previous   SGA   analysis   experiments   have   also   highlighted   the   significance   of   negative   genetic   interactions  over  positive  genetic  interactions  since  virtually  all  of  negative  genetic   interactions   contain   at   least   one   essential   gene   for   a   particular   process   (Baryshnikova   et   al.,   2010).   It   was   concluded   that   negative   genetic   interactions   were   more   reliable   than   positive   genetic   interactions;   therefore   the   latter   was   excluded  from  this  study.    

Chapter  5.  An  unbiased  approach  to  identify  genetic  interactions  of  HSP12/HSP26     hsp12/hsp26∆  double   mutant   identified   and   discussed   in   chapter   3   suggests   that  

HSP12  and  HSP26  genetically   interact.   Disappointingly,   SGA   analysis   performed   in   this   study   did   not   identify   a   genetic   interaction   between  HSP12  and  HSP26.  SGA   analysis   is   a   well-­‐established   technique   for   determination   of   strong   genetic   interactions.   However,   because   of   the   methodology   of   replica   plating   from   solid   cultures,  SGA  analysis  can  sometimes  not  detect  when  there  is  a  slight  reduction  in   the   growth   of   a   double   mutant   in   comparison   to   the   control.   In   contrast,   the   methodology   used   in   QFA   involves   the   dilution   of   solid   cultures   into   liquid   media   thereby   providing   much   more   detailed   fitness   measurements   and   allowing   identification   of   slight   genetic   interactions.   It   will   therefore   be   interesting   to   determine   if   QFA   identifies   a   genetic   interaction   between  HSP12   and   HSP26.  

Alternatively,  it  may  be  that  HSP12  and  HSP26  do  not  genetically  interact  with  one   another  but  interact  with  common  genes.          

  Comparing  the  genetic  interactions  of  HSP26  identified  in  this  study  to  those   identified  by  other  genetic  studies  revealed  only  one  identical  genetic  interaction,  

YDJ1,  that  is  a  consistent  finding  in  this  study  and  a  study  performed  by  Duennwald   et  al,  (Duennwald  et  al.,  2012).  YDJ1  encodes  a  type  1  Hsp40  co-­‐chaperone  involved   in  the  functional  regulation  of  Hsp90  and  Hsp70  (Caplan  and  Douglas,  1991).  Hsp26   has   been   shown   to   interact   physically   with   Hsp104   and   Hsp70   during   the   reactivation   of   proteins   (Haslbeck   et   al.,   2005).   Furthermore,   Hsp104,   Hsp70   and   Hsp40  are  known  to  be  essential  for  yeast  cell  viability  (Xu  et  al.,  2013).  This  result   suggests  that  HSP26  and  YDJ1  interact  genetically  and  may  overlap  functionally  to   some  extent.    

  Comparing  the  genetic  interactions  of  HSP12  identified  in  this  study  to  those   identified  in  other  genetic  studies  did  not  reveal  any  identical  genetic  interactions.   In   this   study   we   identified   genetic   interactions   between  HSP12  and  CDC9  and  

CDC45.   Similar   to   this,   Costanzo   et   al,   reported   a   genetic   interaction   between  

HSP12  and  CDC3  (Costanzo  et  al.,  2010).  CDC3  is  a  component  of  the  septin  ring  and   required   for   cytokinesis   (Takizawa   et   al.,   2000).  CDC9  and  CDC45  are   involved   in   DNA  replication  and  differ  in  functions  to  CDC3  (Willer  et  al.,  1999,  Takizawa  et  al.,   2000,  Tye,  1999).  Although  it  may  appear  that  the  genetic  interactions  are  similar   because   of   the   CDC   gene   class,   this   is   misleading   as   they   actually   perform   very  

Chapter  5.  An  unbiased  approach  to  identify  genetic  interactions  of  HSP12/HSP26    

different   roles   in   the   cell.   This   underlies   the   need   to   do   GO   term   analysis   rather   than  rely  on  gene  class  names  as  an  indicator  of  function.        

  This   study   also   identified   a   similar   genetic   interaction   of  HSP26   to   that   reported   by   Constanzo   et   al.  In   this   study   a   genetic   interaction   was   identified   between  HSP26  and  RPL22A.   RPL22A   encodes   a   protein   component   of   the   large   (60S)   ribosomal   subunit   and   this   result   was   found   to   be   consistent   with   that   reported  by  Costanzo  et  al,  (Venema  and  Tollervey,  1999)  (Costanzo  et  al.,  2010).   Costanzo  et  al.,  reported  HSP26  to  have  a  negative  genetic  interaction  with  RRP1,  

which  encodes  a  protein  necessary  for  biogenesis  of  60S  ribosomal  subunits  (Horsey   et  al.,  2004).  

  There  were  only  a  limited  number  of  identical  genetic  interactions  of  HSP26  

consistent  in  this  study  when  compared  to  the  existing  literature.  Despite  this,  there   were  similarities  in  GO  terms  identified  by  this  study  and  by  other  genetic  studies.   For  example,  a  genetic  interaction  has  been  reported  for  HSP26  and  NPL3  (Moehle   et   al.,   2012).   NPL3   encodes   an   RNA-­‐binding   protein   involved   in   repressing   translation   initiation   and   mRNA   processing   (Windgassen   et   al.,   2004).   This   study   identified   a   genetic   interaction   between  HSP26  and   genes   involved   in   translation   initiation  and  mRNA  processing.    

5.4.2  The  genetic  link  between  the  HSP12  and  HSP26  and  genes  involved  in  stress