Elucida(ng  the  Mechanism  of  Purifying  Selec(on  using  C.  elegans  Adi(  Trivedi,  Sagen  Peterson,  Professor  Joel  Rothman  Molecular,  Cellular  and  Developmental  Biology,  UCSB  

Mitochondria   are   the   main   source   of   ATP   producAon   for   most  cells   through   a   series   of   redox   reacAons.   Thus,   healthy  mitochondria   are   necessary   for   growth   and   survival.  Mitochondrial   DNA   (mtDNA),   encoding   12   proteins   in   these  energy   producing   steps,   follows   a  maternal   inheritance   paJern,  but   has   a   low   generaAonal   mutaAon   rate.   The   low   rate   of  inherited   mtDNA   mutaAons   suggests   the   presence   of   a   strong  mtDNA  selecAon  pressure  in  the  germline.  Purifying  selecAon,  as  this   mechanism   is   called,   ensures   healthy   mtDNA   inheritance,  reducing   chances   of   progeny   with   defecAve   mitochondria.   We  aim   to     study   the   rate   of   purifying   selecAon   with   a   focus   on  germline  apoptosis,  and  to  idenAfy  the  apoptoAc  genes  involved,  beginning  with  ced-­‐3.    

Germline  apoptosis  and  Purifying  Selec(on    During   oogenesis   in  C.   elegans,   >90%   potenAal   oocytes   are  killed  off,   leaving  a  small  subset  of  cells  with  mtDNA  copies,  to  develop  into  mature  oocytes1.  Perhaps  this  selects  against  mutant   mtDNA,   eliminaAng   them   from   the   immortal  germline.  (Fig.  1)    

Q:   How   is   germline   apoptosis   regula(ng  mtDNA  inheritance,  and  is  this  a  key  step  of  purifying  selec(on?  

Drug  Selec(on  for  N2  Strain    In   order   to   measure   the   rate   of   purifying   selecAon   using  qPCR   in  WT   and  mtDNA  mutant   strains,   it   is   necessary   to  develop  a   selecAon  condiAon   to  differenAate  between   the  progeny   that   have   successfully   uptaken   WT   mitochondria  from  the  ones  that  have  not.  We  are  using  a  viable  mtDNA  mutant   strain,   JR3630,   and   the   laboratory   WT   strain,   N2,  and   treaAng   them   with   drugs   targeAng   the   mitochondrial  respiratory  chain  (MRC).      

       

Gene(cs:  MT1522  x  JR3630  cross    

Figure   4:   Plasmid   map   of   C.  elegans   mtDNA   showing  uaDf5  deleAon3    

Figure  1:  Proposed  mechanism  of  purifying  selecAon  during  germline  apoptosis  in  one  gonadal  arm  of  C.  elegans.  

Figure  2:  Chloramphenicol  as  selecAon  condiAon  for  N2  strain  containing  WT  mtDNA    

Table&1:!Summary!of!Drug!Selection!Screen!–!Day!3!Measurements!

Drug! No!Observable!Difference!

Differential!growth!observed!Concentration! Type!

Chloramphenicol! ! 10ug/mL,!500ug/mL,!1mg/mL!

JR3630!shows!L3!arrest!while!N2!are!gravid!adults!

Doxycycline! X! ! !Rotenone! X! ! !Antimycin! X! ! !

Sodium!Azide! ! 10uM! Reduced!Survival!of!JR3630!Oligomycin! X! ! !Paraquat! ! 1mM!

500mM!Reduced!health!and!survival!of!JR3630!

Dinitrophenol!(DNP)!

X! ! !

CuCl2! X! ! !Ethidium!Bromide!

(EtBr)!! 25ug/mL! JR3630!show!slower!rate!of!movement!

This   ced-­‐3(n717) ;   uaDf5//++   double   mutant   has  nonfuncAonal   CED-­‐3,   a   pro-­‐apoptoAc   protein   involved   in  acAvaAon   of   the   germline   apoptoAc   pathway.   We   will  introduce   mitochondria   from   the   laboratory  WT   strain,   N2,  and   observe   levels   of   both   types   of   mtDNA’s   throughout  subsequent   generaAons   using   qPCR,   to   determine   purifying  selecAon  rate  changes  due  to  the  ced-­‐3  mutaAon.    

F i g u r e   6 a :  DiagnosAc   Digest  o f   F 2 ’ s   u s i n g  uaDf5  primers  

Figure   6b:   DiagnosAc   Digest   of   F2’s   using    n717  primers  

All   F2’s   contain   the   299  bp   band   ( F i g .   6 a )  indicaAng   presence   of  the   11   gene   mtDNA  deleAon,   uaDf5.   This   is  c o n s i s t e n t   w i t h  maternal   inheritance  paJerns.    

The   459   bp   band   (Fig.6b)   indicates   amplificaAon   of  the  ced-­‐3  gene  through  PCR.  Sanger  sequencing  will  confirm  the  exact  genotype  and  presence  or  absence  of   the   n717   allele   of   ced-­‐3,   which   contains   a   point  mutaAon.    

 1      2      3      4        5        6      7  

Sodium  Azide  inhibits  cytochrome  c  oxidase  (MRC  Complex  IV)  and  Paraquat  produces  ReacAve  Oxygen  Species  (ROS)2.  Exposure  to  both  drugs  appear  to  effect  the  viability  of  the  JR3630  strain  more  criAcally  than  the  N2  strain.  EtBr  inhibits  mtDNA   replicaAon,   and   treatment   of   25ug/mL   produces  mobility  differences.  The  most  consistent  difference  among  concentraAons   was   seen   with   the   mtDNA   translaAon  inhibitor,  Chloramphenicol  (Fig.  2).    

Molecular  Cloning:    Mitochondrial  GFP  to  be  expressed  during  early  embryo  cell  division  stages  

Insert  +  Bam

H1  

Insert  +  Bam

H1  

Insert  +  Bam

H1  +  AatII  

Uncut  Insert  

Figure  7:    (Above)  In  order  for  mitochondrial  GFP   to   be   transcribed   during   the   desired  early   embryo   cell   division   stages,   its  sequence   must   be   driven   by   the   pie-­‐1  promoter.5    

Figure   8:   (Leh)   RestricAon   digest   of   insert  plasmid.   Lane   4   shows   the   1604   bp   band  consistent   with   mitLS   +   GFP   insert  sequence.    

1.  Gumienny  TL,    Lambie  E,  Hartwieg  E,  Horvitz  RH,  Hengartner  MO.  GeneAc  control  of  programmed  cell  death  in  the  caenorhabdi=s  elegans  hermaphrodite  germline.  (1999).  Development.  126,  1011-­‐1022.    2.    Zeitoun-­‐Ghandour  S,  Leszcyszyn  Ol,  Blindauer  CA,  Geier  FM,  Bundy  JG  and  Sturzenbaum  SR.    C.  elegans  metallothioneins:  response  to  and  defence  against  ROS  toxicity.  (2011)  Mol  Biosyst.  7,  2397-­‐406.  3.  Tsang  WY,  and  Lemire  BD.  Stable  heteroplasmy  but  differenAal  inheritance  of  a  large  mitochondrial  deleAon  in  nematodes.  (2002).  Biochem.  Cell  Biol.  80,  645-­‐654.  4.  Skjeldam  HK,  et  al.  Loss  of  Caenohabdi=s  elegans  UNG-­‐1  uracil-­‐DNA  glycosylase  affects  apoptosis  in  response  to  DNA  damaging  agents.  (2010).  DNA  Repair.  9,  861-­‐870.  5.  Ghosh  D,  Seydoux  G.  InhibiAon  of  TranscripAon  by  the  Caenorhabdi=s  elegans  germline  protein  PIE-­‐1:  GeneAc  Evidence  for  DisAnct  Mechanisms  TargeAng  IniAaAon  and  ElongaAon  

²   Exposure  to  Chloramphenicol  results  in  the  most  significant  and    consistent  selecAon  for  N2  strain  so  far    

Will  conAnue  screen  with  Chloramphenicol  in    combinaAon  with    other  MRC  targeAng  drugs  

²   ProducAon  of  ced-­‐3(n717);  uaDf5//++  double  mutant  through  molecular  geneAcs  cross    

Will  be  used  in  qPCR  measurements  to  track  rate  and  involvement  of  ced-­‐3  gene  in  purifying  selecAon  

²   Plasmid  construct  through  classical  cloning  and  restricAon  digests    

Will  be  confirmed  through  digests,  and  microinjected  into  C.  elegans.    

I   would   like   to   thank   Professor   Joel   Rothman   for   providing   a  unique   and   sAmulaAng   undergraduate   research   opportunity,  and  Sagen  Peterson  for  her  mentorship  and  guidance  in  the  lab.  I  also  would   like   to   thank   the  NIH   for   support,  and   the  Arnold  and   Mabel   Beckman   FoundaAon   for   the   Beckman   Scholars  Award.  

Abstract  

Experimental  Studies  and  Results  

Introduc(on  

Acknowledgements  

Concluding  Remarks  and  Future  Direc(ons  

References  

Figure   5:   CED-­‐3   is   a   pro-­‐apoptoAc   protein   in   the  germline4    

n717  459  bp  −  

uaDf5  299  bp−  

 1          2          3            4          5            6            7    1        2          3          4          5            6            7  

4574  bp−  

1604  bp−  

Figure   3:   (Above)   The   uaDf5   deleAon  mutaAon   follows   maternal   Inheritance  paJerns,     while   the   ced-­‐3   point   mutaAon  follows  a  Mendelian  Inheritance  mode.