I STANBUL TECHNI CAL UNI VERSI TY INS TI TUTE OF SCI ENCE AND TECHNOLOGY
M. Sc. Thesi s by Agne STONKUTE
De part me nt : Advanced Technol ogi es
Progra mme: Mol ecul ar Bi ol ogy – Geneti cs & Bi otechnol ogy
SEPTE MBER 2005
İSTANBUL TECHNI CAL UNI VERSI TY INSTI TUTE OF SCI ENCE AND TECHNOLOGY
M. Sc. Thesi s by Agne STONKUTE
(521031200)
Dat e of sub missi on : 2 Sept e mber 2005 Dat e of defence exa mi nati on: 16 Sept e mber 2005
Supervi sor ( Chai r man) : Assi st. Prof. Dr. Arzu KARABAY KORKMAZ
Me mbers of t he Exa mi ni ng Co mmi ttee Assi st. Prof. Dr. Zeynep Petek ÇAKAR ÖZTEMEL (İ. T. Ü.)
Prof. Dr. Sel ma YI LMAZER (İ. Ü.)
SEPTE MBER 2005
İSTANBUL TECHNI CAL UNI VERSI TY INSTI TUTE OF SCI ENCE AND TECHNOLOGY
YÜKSEK Lİ SANS TEZİ Agne STONKUTE
(521031200)
Tezi n Enstitüye Veril diği Tari h: 2 Eyl ül 2005
Tezi n Savunul duğu Tari h: 16 Eyl ül 2005
Tez Danı ş manı: Yr d. Doç. Dr. Arzu KARABAY KORKMAZ
Di ğer Jüri Üyel eri : Yr d. Doç. Dr. Zeynep Petek ÇAKAR ÖZTE MEL (İ. T. Ü.) Prof. Dr. Sel ma YI LMAZER (İ. Ü.)
EYLÜL 2005
ACKNO WLEDGMENT
I woul d li ke t o t hank my s uper vi sor, Ar zu Kar abay Kor k maz, f or l eadi ng me t hr ough my first lab experi ence i n an opt i mal way: al ways gi vi ng advi ces when necessary but al so l eavi ng so me space for i ndependent deci si ons.
I a m al so gr at ef ul t o all my l ab nei ghbours f or shari ng t heir experi ence and fri ends hi p wi t h me.
I want t o t hank my fa mily f or supporti ng me during my st udi es.
TABLE OF CONTENTS
ABBREVI ATI ONS i vi
LI ST OF TABLES vi
LI ST OF FI GURES vii
ÖZET viiii
SUMMARY i xi
1. I NTRODUCTI ON 1
1. 1. Micr ot ubul es and t heir dyna mi cs 1
1. 2. Kat ani n 5
1. 2. 1. AAA pr ot ei ns 5
1. 2. 2. Struct ure and functi ons of kat ani n 6
1. 2. 3. Kat ani n subunits 11
1. 3. Obj ecti ves of t he research 12
2. MATERI ALS AND MET HODS 14
2. 1. Mat eri als 14
2. 1. 1. Equi p ment 14
2. 1. 2. Che mi cal s and enzy mes 14
2. 1. 3. Buffers 16
2. 1. 4. Speci al reagent s and kits 17
2. 1. 5. Bact eri al strai ns 17
2. 1. 6. Bact eri al cult ure medi a 17
2. 1. 7. T/ A cl oni ng vect or wit h cl oned i nsert 18
2. 1. 8. Expr essi on vect or 18
2. 1. 8. 1. pET expressi on syst e m 18
2. 1. 8. 2. pET - 30a vect or 19
2. 2. Met hods 21
2. 2. 1. Small scal e pl as mid DNA preparati on ( mi ni -prep) 21 2. 2. 2. Det er mi nati on of nucl ei c aci d concentrati on 22 2. 2. 3. Preparati on of chemi call y co mpet ent cells – cal ci u m chl ori de met hod 22
2. 2. 4. Transf or mati on of co mpet ent cells 23
2. 2. 5. DNA cl eavage with restri cit on endonucl eases 24
2. 2. 6. Li gati on 25
2. 2. 7. Agar ose gel el ectrophor esi s 26
2. 2. 8. Isol ati on of DNA frag ment s fro m agar ose 27
2. 2. 9. DNA sequenci ng 28
2. 2. 10. Ali gn ment of sequences 29
2. 2. 11. Pr ot ei n expressi on i nducti on 29
2. 2. 12. Cell fracti on analysi s 29
2. 2. 12. 1. Tot al cell pr ot ein anal ysi s 29
2. 2. 12. 2. Sol ubl e t ot al cell pr ot ei n anal ysi s 29 2. 2. 13. El ectrophor esi s of pr ot ei ns on SDS- pol yacril a mi de gel s 30
2. 2. 14. Met al affi nit y purifi cati on of 6xHi s t agged p60- kat ani n 32
2. 2. 15. West er n bl ot 34
2. 2. 16. Bradf or d pr ot ei n det er mi nati on assay 36
3. RESULTS 38
3. 1. Cl oni ng of p60- kat ani n 38
3. 2. Expr essi on st udi es 40
3. 3. Purifi cati on of p60- kat ani n under nati ve condi ti ons 43
3. 4. West er n bl ot anal ysis 44
4. DI SCUSSI ON 46
4. 1. Sequenci ng of t he pET30- p60 construct 46
4. 2. SDS- PAGE anal ysi s of t ot al pr ot ei n fracti on 46
4. 3. Fact ors affecti ng solubilit y of expr essed p60-kat ani n 46
4. 4. Purifi cati on of p60- kat ani n 49
5. CONCLUSI ONS 50
REFERENCES 51
APPENDI X 57
ABBREVI ATI ONS
A2 80 : Absor bance at 280 n m
AAA : ATPases associ at ed with vari ous cell ul ar acti vities ADP : Adenosi ne di phosphat e
AMPS : Ammoni u m persul phat e ATP : Adenosi ne tri phospahe AP : Al kali ne phosphat ase
BCI P : 5- br o mo- 4-chl or o- 3-i ndol yl phosphat e BLAST : Basi c Local Ali gn ment Search Tool
bp : Base pair
BPB : Br o mophenol bl ue BS A : Bovi ne ser u m al bu mi n CBB : Co massi e Brilliant Bl ue
EDTA : Et hyl enedi a mi net etraaceti c aci d E- sit e : Exchangeabl e sit e
GTP : Guanosi ne tri phospaht e Hi s : Histi di ne
I MAC : I mmobili zed- met al affinit y chr o mat ography I PTG : Isopr opyl- b D- t hi ogal act opyr anosi de
kb : Kil obase
k Da : Kil odalt on
LB medi a : Luri a Bert ani medi a
MAP : Mi cr ot ubul e associ at ed pr ot ei n NBT : Nitrobl ue t etrazoli um
Ni - NTA : Nickel- nitril otri aceti c aci d OD : Opti cal densit y
PCR : Pol y mer ase chai n reaction
pET : Pl as mi d expressi on by T7 RNA pol y mer ase PVDF : Pol yvi nyli dene difl uoride me mbr ane
RNase : Ri bonucl ease
SDS : Sodi u m dodecyl sulfat e
SDS- PAGE : Sodi u m dodecyl sulfat e pol yacr yl a mi de gel el ectrophor esi s TAE : Tris acet at e EDTA
TBS : Tris buffered sali ne TCA : Tri chl or oaceti c aci d
Tri s : Hydr oxy met hyl a mi nomet hane TTBS : TBS cont ai ni ng Tween20 w/ v : Wei ght/ vol u me
LI ST OF TABLES
Page No
LI ST OF FI GURES
Page No
Fi gure 1. 1: A. a mi cr ot ubul e struct ure. B. -t ubuli n ri ng nucl eati ng
mi cr ot ubul e ( Al berts et al., 2002) …... 1 Fi gure 1. 2: Mo del f or t he struct ural consequences of GTP hydr ol ysi s i n
t he micr ot ubul e l atti ce (Al berts et al., 2002) ……... 2 Fi gure 1. 3: Mo del of conf or mati onal change of AAA pr ot ein ri ng. ( Val e,
2000) ………... 6 Fi gure 1. 4: Mo del f or mi cr ot ubul e severi ng by kat ani n. ( Onl y a si ngl e
pr ot ofil a ment of mi cr ot ubul e i s sho wn). I n t he t op-ri ght part, you see kat ani n- ATP oli go meri zed on mi cr ot ubul e. I n t he bott om-ri ght part, as a r esult of ATPase acti vit y kat ani n ri ng changes conf or mati on l eadi ng t o mechani cal strai n. I n t he bott om-l eft part, t ubulin-t ubuli n bonds ar e broken, kat ani n di ssoci at es fr o m t he co mpl ex. I n t he t op-l eft part, kat ani n- ADP co mpl ex i s r eady t o exchange ADP f or ATP and j oi n t he cycl e
agai n. ( Hart man & Val e, 1999) ……….. ………. … 7 Fi gure 1. 5: Effect of mi cr ot ubul e concentrati on on kat ani n’s acti vit y
( Hart man and Val e, 1999) ……….. . 9
Fi gure 1. 6: Model of kat ani n’s r ol e i n t he mit otic spi ndl e. Severi ng of t he
ki net ochor e mi cr ot ubul e by kat ani n r el eases a s hort mi cr ot ubul e frag ment ( A- B) and all ows t he ki nesi n-li ke pr ot ei n t o pull t he ki net ochor e mi cr ot ubul e t o war d t he centr oso mal mat ri x ( B). Si mult aneous pol y meri zati on of
t ubuli n occurs at t he pl us end ( B- C). ( Mc Nall y et al., 1996)... 10 Fi gure 1. 7: Ratt us norvegi cus p80-kat ani n a mi no aci d sequence ( Gene
Bank, t he Eur opean Mol ecul ar Bi ol ogy Labor at ory, accessi on No. Q8 BG40). WD motifs are s ho wn i n bol d conser ved
di pepti de sequences are underli ned ………. 11 Fi gure 1. 8: Ratt us norvegi cus p60-kat ani n a mi no aci d sequence ( Gene
Bank, t he Eur opean Mol ecul ar Bi ol ogy Labor at ory, accessi on No. AY621629). Part of p60 s ho wn i n bol d i s t he sequence whi ch was cl oned and expr essed; underli ned part of p60 i s t he
conser ved C-t er mi nal AAA do mai n ……… 12
Fi gure 2. 1: Vect or map of pCR2. 1- TOP O wi t h cl oned i nsert. Cl oni ng/restri cti on area wi t h sche mati call y r epresent ed i nsert is sho wn i n a l ar ger scale. Arr ow stret chi ng fr o m PCR pr oduct poi nt s at t he part of cl oned p60 s ubunit ( begi nni ng and end of t he sequence) i n a l ar ge scal e. At t he bott om-l eft part of t he
Fi gure 2. 2: Vect or map of pET30a. Sour ce:
htt p:// www. e mdbi osci ences. co m... 20 Fi gure 2. 3: Cl oni ng/ expressi on regi on of t he codi ng strand of pET30a.
Sour ce: htt p:// www. e mdbi osci ences. co m... 20 Fi gure 2. 4: I midazol e ri ng struct ure and histi di ne ………. 33 Fi gure 3. 1: Sche me of p60 s ubcl oni ng. P60- kat ani n i s cut wi t h Hi nd III
and Eco RI fr o m PCR2. 1 – TOP O vect or (l eft above) and subcl oned i nt o pET- 30a expr essi on vect or (ri ght above). Res ulti ng construct where p60 i s f used t o Hi s-t ag i s sho wn at t he bott om of t he pi ct ure. p60 sequence part i s writt en i n bi g lett ers; it changes ori ent ati on aft er subcl oni ng. A part of PCR2. 1 – TOP O vect or sequence cont ai ni ng stop codon i n
non- codi ng strand is written i n s mall l ett ers ……….. . 38 Fi gure 3. 2: Restri ct ed i nsert and expr essi on vect or DNAs ext ract ed fr o m
agar ose gel and r un on a gel agai n. Lane 1 - pET30a vect or restri ct ed wi t h R buffer, Lane 2 – pET30b vect or r estri ct ed wi t h Y buffer, Lane 3 – p60 r estri ct ed fr o m p CR 2. 1- TOP O wi t h R buffer, Lane 4 – p60 r estri ct ed fr o m p CR 2. 1- TOP O
wi t h Y buffer (poi nt ed by arr ow) ……… 39 Fi gure 3. 3: Restri ct ed purifi ed transfor mant pl as mi ds. All t hr ee sa mpl es
(l anes 1, 2, 3) are cut t o pr oduce t wo seg ment s: one i n t he
regi on bet ween 5- 6 kb and anot her i n t he regi on of 600 bp …… 40 Fi gure 3. 4: SDS- PAGE anal ysi s of t ot al pr ot ei n sa mpl es ( bands of
over-expr essed pr ot ei n are point ed by t he arrows) ……….. 41 Fi gure 3. 5: SDS- PAGE anal ysi s of sol ubl e and i nsol ubl e fracti ons ……….. 42 Fi gure 3. 6: SDS- PAGE anal ysi s of purifi ed p60- kat ani n ……….. 43 Fi gure 3. 7: We st er n bl ot anal ysi s of t ot al pr ot ei n fracti on and purifi ed
pr ot ei n ……….. 45
Fi gure 4. 1: Rat p60- kat ani n a mi no aci d sequence. Non- pol ar (hydr ophobi c) a mi no aci ds ar e r epl aced by ast erisks. Cyst ei n i s mar ked i n gr ey. Expr essed sequence i s sho wn i n bol d. C – ter mi nal conser ved do mai n i s underli ned. Ext ensi ve
hydr ophobi c regi ons are poi nt ed by arrows ……… 47 Fi gure B. 1: Al i gn ment results. Expr essi on vect or part is underli ned, T/ A
vect or part is i n bol d, t he rest is p60 part ……… 57 Fi gure B. 2: pET 30a- p60 sequenci ng results ………. 58
P60- KATANİ N’ İ N KLONLA MASI VE EKSPRES YONU
ÖZET
Kat ani n mi kr ot übüll eri parçal ayan bir pr ot ei ndir. 60 ve 80 k Da’l uk poli pepti dl erden ol uşan bu het erodi meri k pr ot ei n, st abil mi kr ot übüll eri parçal ayabil mek içi n ATPye i hti yaç duyar. Kat ani ni n bi yol oji k r ol üne geli nce, bu pr otei ni n mit ozda öne mli dir. Kat ani n, mit ozda mi kr ot übüll eri n eksi uçları nı n bağlı ol dukl arı sentrozo ml ar dan ayrıl ması na aracı ol makt adır ve böyl ece mi kr ot übüll ere bağl anmış ol an kr o mozo ml arı n akışı sağl anmakt adır. Mi kr ot übüll eri n sentrozo ml ar dan ayrıl ması sinir hücrel eri nde çok öne mli dir. Ser best mi kr ot übüll er uzayan aksonl ara t aşı nmakt adır ve burada akson i çi n gerekli ol an fi zi ksel dest eği sağl a makt adırlar. Mikr ot übüll eri n di ğer bir görevi de or ganelleri n t aşı nması nda sübstrat vazifesi gör mektir.
Kat ani n i ki alt ünit eden ol uş makt adır, onl arı n i si mleri mol ekül er ağı rlı kl arı na gör e belirlenmiştir. Kat ani ni n alt ünit el eri ni n araştırılması bu enzi min akti vitesi ni n ve r oll eri ni n daha i yi anl aşıl ması nı sağl ayacaktır. p80 ve p60 anti korl arı bu araştır malar da kull anıl acaktır. Monokl onal anti kor poli kl onal anti kora t erci h edil mekt edir, çünkü monokl onal anti kor neredeyse bit meyen bir anti kor kaynağı dır ve t ek bir epit op t anı yabil me özelli ği ne sahi ptir. p60’ı n ekspresyonu gerçekl eştiril miş ol ması na r ağ men, pr ot ei n çözün müş hal de el de edile me mi ştir. Bu dur um monokl onal anti kor üretimi ni zorlaştır makt adır.
Bu çalış mada küçük (600 kb) özel bir p60-kat ani n kı s mı ekspresyon vekt ör üne kl onl anarak ekspresyonu ger çekl eştiril di. Ekspr es edil en pr ot ei n çözün müş hali nde bul un makt adır. Sonuçt a üretilen çözün müş pr ot ei n i mmobilize met al afinit e kr omat ografisi yönt e mi yl e doğal şartlar altı nda saflaştırıl dı.
Bu pr oj eni n sonraki aşamal arı nda da reko mbi nant p60- kat ani ni kull anılarak monokl onal anti kor üretilecektir. Ür etilen anti kor un p60 kat ani n i nhi bisyonu, p60’ kat ani ni n ekspresyonunun ve l okalizasyonun i n vi vo gözl en mesi gi bi pek çok geni ş kull anı m al anı ol acaktır. Bu çalış mal ar mi kr ot übüll er ve kat ani nl e il gili hast alı kl arı nı n anl aşılabil mesi ni sağl ayacaktır ve hatta bel ki uzun vadede bu hast alı kl arı n i yileştiril mesi nde kull anılacaktır.
P60- KATANI N CLONI NG AND EXPRESSI ON
SUMMARY
Kat ani n i s a mi cr ot ubul e severi ng pr ot ei n. Thi s het er ody meri c pr ot ei n ( 60 and 80k Da pol ypepti des) r equires ATP t o sever and di sasse mbl e st abl e mi cr otubul es. Kat ani n i s i mport ant i n mit osi s, it me di at es t he di sasse mbl y of mi cr ot ubul e mi nus ends duri ng pol e war d fl ux. Rel ease of mi cr ot ubul es fr o m centr oso me i s al so essenti al i n neur onal cells. Rel eased mi cr ot ubul es are transport ed i nt o gr owi ng axons wher e t hey pr ovi de archi tect ural s upport and a substrat e al ong whi ch organell es are transport ed i n bot h directi ons wit hi n t he axon.
Kat ani n consi sts of t wo s ubunits: p60 and p80 whi ch wer e na med accor di ng t o t heir mol ecul ar wei ght. St udi es of kat ani n’s subunits woul d enabl e us t o underst and acti vit y and rol es of kat ani n bett er. Anti bodi es agai nst p80 and p60 wi ll be used i n t hese st udi es. Mo nocl onal anti bodi es are pr eferred agai nst pol ycl onal anti bodi es si nce t hey ar e an al most i nfi nit e sour ce of antibodi es and r ecogni ze one si ngl e epit ope. Ho wever, expr essi on experi ment s wit h p60 r esult ed i n an i nsol ubl e pr ot ei n until no w and t hi s hi nders pr oducti on of monocl onal anti body.
In t hi s st udy a s hort specifi c sequence ( 600 kb) of p60- kat ani n was expr essed aft er bei ng cl oned i nt o expr essi on vect or. Expr essed pr ot ein i s f ound i n t he s ol ubl e fracti on. Fi nall y, sol ubl e pr ot ei n was purified usi ng met al affi nit y chr o mat ography under nati ve conditi ons.
The next st ep will be t o pr oduce monocl onal anti body usi ng r eco mbi nant p60- kat ani n. Pr oduced anti body wi ll have wi de appli cati on ar ea e. g. i nhi biti on of p60 subunit, obser vati on of p60 i n vi vo expr essi on and l ocali zati on whi ch wi ll hel p t o underst and hu man di sor ders rel at ed t o mi cr ot ubul es and kat ani n, and maybe event uall y even wi ll be even used t o t reat t he m.
1. I NTRODUCTI ON
1. 1. Mi crot ubul es and t hei r dyna mi cs
Mi cr ot ubul es are pol ymers made of repeati ng α/ β-t ubuli n het erodi mers. I n cells, t hey are us uall y or gani zed i nt o 13 li near pr ot ofil a ment s t o f or m a cyli ndri cal str uct ure. Mi cr ot ubul es are nucl eat ed fr o m -t ubuli n cont ai ni ng ri ng str uct ures wi t hi n t he centr oso mes (see Fi g. 1. 1).
A B
Fi g. 1. 1: A. a mi cr ot ubul e struct ure. B. -t ubuli n ri ng nucl eati ng mi cr ot ubul e. ( Al berts et al., 2002)
Mi cr ot ubul es are nucl eated at t he centr oso me at t heir mi nus ends, so t he pl us ends poi nt out war d and gr ow t o war d t he cell peripher y. Minus end gr ows sl ower co mpared t o t he pl us end and mi nus end i s t er mi nat ed by α t ubuli n subunits, whil e pl us end i s t er mi nat ed by β s ubunits. Micr ot ubul es are i mport ant i n mai ntai ni ng cell shape, i n cell transport, cell motilit y and cell di vi si on.
Mi cr ot ubul es ar e dyna mic struct ures, t hey can gr o w or s hri nk by addi ng or l oosi ng subunits at t heir ends ( at t he pl us end pri maril y). Thi s ki nd of dyna mi cs i s based on t he bi ndi ng and hydr ol ysis of GTP by t ubuli n subunits. The body of micr ot ubul e ma de of GDP-t ubuli n subunits i s unst abl e (see Fi g. 1. 2). Tubuli n di mers at t he ends still ret ai n t heir GTP and st abili ze mi cr ot ubule. When t hi s GTP cap i s l ost, mi cr ot ubul e depol y meri zes ( Nogal es, 2000; Ho ward and Hy man 2003).
Fi g. 1. 2: Model f or t he struct ural consequences of GTP hydr ol ysi s i n t he mi cr ot ubul e
latti ce. ( Al berts et al., 2002)
Such behavi or i s based on t he bi ndi ng and hydr ol ysi s of GTP at t he nucl eoti de exchangeabl e sit e ( E-site) on β- t ubuli n. Onl y di mers t hat have GTP i n t heir exchangeabl e E-sit e can pol y meri ze, but f oll owi ng pol y meri zati on t hi s nucl eoti de i s
hydr ol yzed and beco mes non- exchangeabl e ( Heal d and Nogal es, 2002; Ga dde and Heal d, 2004).
Si nce t ubuli ns are pr eferenti all y i ncor por at ed at t he pl us end, and mor e r api dl y l ost at t he mi nus end, a uni directi onal fl ux, or tread milli ng, of t ubuli n s ubunits al ong t he axi s of micr ot ubul e arises ( Mai at o et al., 2004).
De pol y meri zati on, repol y meri zati on, tread milli ng char act eristi cs can be r egul at ed by mi cr ot ubul e associ at ed prot ei ns ( MAPs). Tr aditi onall y, a pr ot ei n was consi dered t o be a MAP i f it coul d be co- purifi ed i n vitro wi th mi cr ot ubul es as a r esult of direct bi ndi ng ( Mai at o et al., 2004, Sedbr ook 2004). MAPs wer e s ho wn t o sti mul at e mi cr ot ubul e asse mbl y. Subsequentl y, it was pr oposed t o defi ne MAPs as mi cr ot ubul es bi ndi ng pr otei ns i n vi vo. No w, t hey are usuall y defi ned as prot ei ns t hat bi nd t o mi cr ot ubul es and change t heir st abilit y and mechani cal pr operti es. MAPs ar e not r estri ct ed onl y t o stabili ze mi cr ot ubul es, some can medi at e t he i nteracti on of mi cr ot ubul es wit h ot her cell ul ar co mponent s and s o me can dest abili ze or sever e mi cr ot ubul es. MAPs can act on a mi cr ot ubul e directl y, or t hey can r estri ct access t o t he mi cr ot ubul es t o ot her MAPs or mot or pr ot ei ns by bi ndi ng t o mi cr ot ubul e ( Baas & Qi ang, 2005). A br oad r ange of MAPs f uncti ons s uggest s t hat it i s t he coor di nat ed acti on of MAPs t hat l eads t o t he pr oper mi cr ot ubul e f uncti oni ng ( Mai at o et al., 2004). Coor di nati on f aul ts may l ead t o di seases, e. g. Al zhei mer’s ( Baas & Qi ang, 2005).
Wh y do mi cr ot ubul es need t o be dyna mi c? Microt ubul es ser ve as hi ghwa ys al ong whi ch car goes ar e transport ed i n a cell (t hey can move vari ous cell cont ent s vi a speci al att ach ment pr ot eins), t hey pr ovi de struct ural support f or t he cell and or gani ze me mbr anous or ganell es. In or der t o f ulfill t hese functi ons, mi cr ot ubul es must f oll ow t he cell cycl e, t he cell gro wt h and r earrange t he msel ves adapti ng t o t he cell needs at parti cul ar mo ment.
One exa mpl e of mi cr ot ubul es’ r ol e requiri ng dyna mi c pr operties i s in mit oti c spi ndl e. The s pi ndl e consi sts of mi cr ot ubul es as well as ot her pr ot ei ns t hat alt er mi cr ot ubul e dyna mi cs. Mi cr ot ubul es have t o gro w f r o m separat ed cent ros o mes and capt ure chr o mos o mes i n or der t o f or m a s pi ndl e. One model of such asse mbl y, ‘ ‘search and capt ure model’ ’ says t hat mi cr ot ubul es e manati ng from a centroso me under go cycl es of gr owt h and shri nkage, randoml y pr obi ng t he cyt opl as m until runni ng i nt o a ki net ochor e, wit h whi ch t hey f or m a st abl e att achme nt. When chr o mos o mes segregat e, t hey ar e carri ed t o t he di sti nct pol es by short eni ng
mi cr ot ubul es. Anot her model, ‘ ‘pack man’ ’, pr oposes t hat t he ki net ochore i nduces mi cr ot ubul e di sasse mbl y at t he pl us-ends, but mai nt ai ns att ach ment as t he fi ber depol y meri zes, t hus chewi ng it s way t o t he pol e. The ot her model ‘ ‘tracti on fi ber’ ’ pr oposes t hat pol e- war d mi cr ot ubul e fl ux i s harnessed t o move t he chro mos o me. Act uall y, bot h mechani sms wor k ( Gadde and Heal d, 2004).
Mi cr ot ubul es have an i mport ant r ol e i n s pecifi c acti viti es of differenti ated cell s. I n neur ons, bundl es of mi crot ubul es stret ch fr o m t he cell body t o t he ti ps of el ongat ed neur al pr ocesses kno wn as axons and dendrit es. Axon can be a met er l ong, s o i f mi cr ot ubul es woul d be att ached t o centr oso me, t hen one mi cr ot ubul e shoul d be stret chi ng f or a met er. Ho wever, i n neur onal cells (as well as i n ot her asy mmet ri c differenti at ed cells) mi crot ubul es are not att ached t o t he centr oso me ( Keat i ng et al., 1997) and no one mi cr otubul e stret ches t he entire l engt h of t he axon; i nstead, short overl appi ng seg ment s of parall el mi cr ot ubul es ma ke t he el ongat ed mi cr ot ubul e struct ures. Micr ot ubul es pr ovi de s upport f or t he gr owt h and mai nt enance of t he pr ocess and al so pr ovi de a s ubstrat e al ong whi ch or ganell es are transported i n bot h directi ons wi t hi n t he axon ( Ah mad and Baas, 1995; Baas, 2000). Axonal transport of or ganell es, RNA and pr otei n al ong t he mi cr ot ubul es i s absol ut el y r equired for healt h of neur ons, and s o me hu man di seases ( e. g. neuronal degenerati on i n mot or neur on di sease) have t heir bases i n f ault y or bl ocked axonal transport ( Murray & Wol koff, 2003).
In bot h above menti oned exa mpl es, abilit y of microt ubul e struct ures t o move and rearrange t he ms el ves was essenti al f or t heir f uncti oni ng. Ho wever, t hese dyna mi c abiliti es are not onl y r estri ct ed t o i ntri nsi c dynami c charact eristi cs of mi cr ot ubul es. In bot h cases, severi ng of mi cr ot ubul es and r elease of t heir mi nus ends pl ay an essenti al r ol e. Ot her exampl es wher e severi ng of mi cr ot ubul es pl ay an i mport ant r ol e i ncl ude degr adati on of sper m axone mal mi cr ot ubul es aft er f ertili zati on of sea ur chi n oocyt es, mi cr ot ubul e r eorgani zati on duri ng t he t ransiti on fr o m i nt er phase t o mit osi s i n di vi di ng cells, defl agell ati on i n Chl amydo monas ( Lohr et et al., 1998, Quar mby & Lohr et, 1999). I n additi on, severi ng fil a ment s changes t he physi cal and mechani cal pr operti es of t he cyt opl asm: stiff, l ar ge bundl es and gel s beco me mor e fl ui d when t he fil a ment s are severed.
The mi nus ends appearing aft er severi ng wer e never seen t o gr ow and once havi ng begun t o s hort en wer e not obser ved t o r et ur n t o a st abl e st at e, differentl y fr o m t he pl us end. It i s supposed t hat t he mi nus end i s pr otect ed by s o me s pecifi c fact or. So,
cells may pr oduce, rel ease, st abili ze and t ransport centr oso mal mi cr otubul es t o pr oduce non- centr oso mal arrays li ke neur ons ar e doi ng, or cells ma y r el ease centr oso mal mi cr ot ubul es and di sasse mbl e at t he mi nus end t o co mpl e ment pl us end dyna mi c i nst abilit y when r api d r eor gani zati on of mi cr ot ubul e cyt oskel et on i s needed, e. g. duri ng transiti on from i nt er phase t o mit osi s (Keati ng et al., 1997).
1. 2. Kat ani n
1. 2. 1. AAA prot ei ns
Kat ani n bel ongs t o t he AAA s uperfa mil y. AAA ATPases ( ATPases Associ at ed wi t h vari ous cell ul ar Acti vities) pl ay i mport ant r ol es i n nu mer ous cell ul ar acti viti es i ncl udi ng pr ot eol ysi s, prot ei n f ol di ng, me mbr ane t raffi cki ng, cyt oskel et al regul ati on, or ganell e bi ogenesi s, DNA r epli cati on and i ntracell ul ar motilit y. The uni f yi ng feat ure of t he AAA s uperfa mil y i s an ATP- ase do mai n of about 220 a mi no aci ds. It i ncl udes t he Wal ker si gnat ure sequences of P-loop ATPases and ot her r egi ons of si mil arit y uni que t o AAA pr ot ei ns. The cl assi cal AAA pr ot ei ns are easil y recogni zed by t heir strong sequence conser vati on i n t hi s do mai n ( about 30 % i dent it y) ( Val e, 2000; Pat el & Latt eri ch, 1998; Hart man et al., 1998).
AAA pr ot ei ns f uncti on as oli go mers ( Val e, 2000), alt hough accor di ng t o Pat el & Latt eri ch ( 1998) t here are al so mono mers. I n most cases oli go mers f or m hexa meri c ri ngs ( Val e, 2000). Katani n exi sts i n an equili bri um bet ween monomer s and oli go mers. Its oli go meri c st at e has been sho wn t o be a hexa meri c ri ng ( Har t man and Val e, 1999).
Ho w do AAA pr ot ei ns use t he ri ng str uct ure? ATP bi ndi ng i nduces str uct ural rearrange ment s at t he i nterface r egi on whi ch i ncreases i nt eracti ons bet ween adj acent AAA do mai ns as well as bet ween t he AAA pr ot ein and it s t ar get. Thi s cr eates a t ense st at e of t he AAA-t ar get pr ot ei n co mpl ex. The ti ght er subunit-subunit i nt eracti ons i n t ur n accel erat e ATPase reacti on. Once t he AAA modul es are i n ADP st at e, t he co mpl ex r everts t o a r elaxed confi gurati on i n whi ch i nt eracti ons bet ween AAA do mai ns and t he t ar get prot ei n weaken. Ri ngs al so pr ovi de a fra me wor k f or bi ndi ng target pr ot ei n at multi pl e sit es. If t he ri ng- bi ndi ng sit es change t heir positions duri ng t he ATPase cycl e, t hen t ensi on coul d be appli ed t o a bound pr ot ei n (see Fi g. 1. 3. ) ( Val e, 2000).
Fi g. 1. 3.: Model of conf or mati onal change of AAA pr ot ei n ri ng. ( Val e, 2000)
1. 2. 2. St ruct ure and f uncti ons of kat ani n
Kat ani n i s one of t he MAPs and it severes mi cr ot ubul es. It was first purifi ed fr o m s ea ur chi n and charact eri zed i n 1993 by F. J. Mc Nally and R. V. Val e. They na med t he purifi ed pr ot ei n upon ‘ ‘kat ana’ ’, t he Japanese wor d f or sa mur ai s wor d. Kat ani n i s a het er odi meri c pr ot ei n ( 60 and 80k D pol ypepti des). It was t he first pr ot ei n f ound whi ch r equired ATP t o sever and di sasse mbl e st abl e mi cr ot ubul es ( Mc Nal l y & Val e, 1993). Lat er anot her pr otei n, spasti n ( Hazan et al., 1999) whi ch i s r el at ed t o kat ani n, di spl ays ATPase acti vity and uses ener gy fr om ATP hydr ol ysi s t o sever and di sasse mbl e mi cr ot ubul es li ke kat ani n ( Roll- Mecak & Val e, 2005; Evans et al., 2005) was i dentifi ed.
Kat ani n br eaks mi cr ot ubul es al ong t heir l engt h; it does not t ake a way t he t ubuli n di mers at t he end of t he mi cr ot ubul e, but r emoves t he m fr o m t he wall of t he mi cr ot ubul e. Pr oduct of severi ng acti vit y i s t ubul i n di mers. Rel eased t ubuli n di mers are abl e t o r epol y meri ze agai n, so t hey ar e not phosphor yl at ed or ot her wi se changed. Kat ani n can di sasse mbl e mi cr ot ubul es under conditi ons t hat favor spont aneous asse mbl y of t ubuli n i nto mi cr ot ubul es. Thi s means t hat eit her kat ani n i nhi bits pol y meri zati on of t ubulin di mers or t hat kat anin di ssoci at es t ubuli n di mer s fr o m mi cr ot ubul es fast er t han they re-associ at e ( Mc Nally & Val e, 1993).
Tubuli n subunits are hel d i n pl ace t hr ough bot h l ongit udi nal and l at eral contact s, t hey di ssoci at e ver y sl owl y ( 10- 8s- 1) fr o m t he mi cr ot ubul e wall. Severi ng of a microt ubul e
at a s pecifi c spot al ong mi cr ot ubul e l engt h r equires t hirt een s ubunits ar ound t he circu mf erence of t he t ubul e each t o be di ssoci ated fr o m ti ghtl y bound nei ghbours above, bel ow and on t wo si des. Ho wever, i n t he pr esence of ATP, kat ani n pert ur bs t hese t ubuli n--t ubuli n cont act s and can sever and di s mantl e a t axol-st abili zed mi cr ot ubul e wit hi n a coupl e of mi nut es ( Val e, 2000). Micr ot ubul es act as a scaff ol d for kat ani n t o oli go merize aft er it has exchanged it s ADP f or ATP ( see Fi g. 1. 4.). Once a co mpl et e kat ani n ri ng i s asse mbl ed on t he mi cr ot ubul e, t he ATPase acti vit y of kat ani n i s sti mul at ed. As a consequence of ATP hydr ol ysi s and subsequent phosphat e r el ease, t he kat ani n under goes a conf or mati onal change l eadi ng t o mechani cal strai n t hat dest abili zes t ubuli n-t ubuli n cont act s. The ADP- bound kat ani n has l ower affi nit y bot h f or ot her kat ani n mol ecul es and f or t ubuli n; t hi s l eads t o t he di ssol uti on of t he co mpl ex and t he r ecycli ng of t he kat ani n ( Hart man & Val e 1999; Quar mby, 2000; Mc Nally, 2000).
Fi g. 1. 4: Model f or mi crot ubul e severi ng by kat ani n. ( Onl y a si ngl e pr ot ofil a ment of
mi cr ot ubul e i s sho wn). In t he t op-ri ght part, you see kat ani n- ATP oli gomeri zed on mi cr ot ubul e. I n t he botto m-ri ght part, as a r esult of ATPase acti vit y kat ani n ri ng changes conf or mati on l eadi ng t o mechani cal strai n. I n t he bott o m-l eft part, t ubuli n-t ubuli n bonds are br oken, kan-t ani n di ssoci an-t es fr om n-t he co mpl ex. I n n-t he n-t op-l efn-t parn-t, kat ani n- ADP co mpl ex i s r eady t o exchange ADP f or ATP and j oi n t he cycl e agai n. ( Hart man & Val e, 1999).
Mi cr ot ubul e bi ndi ng site f or kat ani n hexa mer i s unkno wn. Possi bl e bi ndi ng sit es i ncl ude t he out si de of t he mi cr ot ubul e, t he mi cr ot ubul e l u men, or t he si des of t ubuli n di mers exposed by hol es i n t he l atti ce ( Mc Nally, 2000; Davi s et al., 2002). It i s
t hought t hat defect s i n t he mi cr ot ubul e l atti ce mi ght ser ve as sit es f or kat ani n acti vit y ( Davi s et al., 2002; Wat er man- St orer and Sal mon, 1997). Kat ani n expl oits l ocal defect s and pr o mot es l oss of t ubuli n at t he defect sit e until t he t wo micr ot ubul e seg ment s are hel d t oget her so weakl y t hat mechani call y unconstrai ned mi cr ot ubul es ki nk at t he def ect sit e ( Davi s et al., 2002). The accel erati on of br eaki ng wi t h i ncreasi ng free ener gy of cur vi ng was f ound i n t he st udy of Odde et al. ( 1999). Ho wever, expect ed degr ee of accel erati on as a f uncti on of el asti c ener gy i ncr ease di d not act i n a pr edi ct able manner. One can i nt er pret t he r esults assu mi ng t hat i ncreasi ng cur vat ure i ncreases MAP di ssoci ati on, whi ch i n t ur n l owers t he ri gi dit y and makes t he mi cr ot ubul e mor e accessi bl e t o severi ng enzy mes. Ot her possi bl e mechani s ms f or t he curvat ure-sensiti vit y i ncl ude cur vat ure-sensiti vit y of severi ng pr ot ei ns and/ or t he t ubuli n di mers t he ms el ves. I ncreased cur vat ure, caused by mi cr ot ubul e mot ors or mechani cal def or mati on of t he cell coul d i ncrease t he nu mber of mi cr ot ubul e def ect s, in t ur n i ncreasi ng t he number of kat ani n severi ng l ocati ons ( Davi s et al., 2002; Odde et al., 1999).
ATPase and severi ng act i viti es of kat ani n are cl osel y r el at ed, but t hey ar e not ti ghtl y coupl ed. ATPase acti vity i s sti mul at ed by mi crot ubul es, whil e i n t he absence of mi cr ot ubul es ATPase act i vit y i s not obser ved. Moreover, mi cr ot ubul es t hat cannot be di gest ed ( e. g. subtilisi n di gest ed ones) sti mul at e ATPase acti vit y. I n t he l att er case, mi cr ot ubul es are not severed or di sasse mbl ed but obser ved kat ani n’s acti vit y was t he sa me as i f mi cr ot ubul es woul d be di gest ed. Thi s sho ws t hat ATPase acti vit y i s not ti ghtl y coupl ed t o severing. When ADP has been used t o i nhi bit ATPase acti vit y, severi ng acti vit y was i nhi bit ed as well whi ch s hows t hat ATPase acti vit y i s necessar y for severi ng ( Mc Nall y and Val e, 1993).
Kat ani n di spl ays an unusual mi cr ot ubul e-sti mul ating r eacti on. ATPase activit y peaks at a mi cr ot ubul e concentrati on of 2 t o 10 mi cro mol s and t hen decreases as t he mi cr ot ubul e concentration i s f urt her i ncreased ( see Fi g. 1. 5). Thi s di ffers fr o m expect ed behavi our accor di ng t o Michaelis- Ment en l a w. There i s t he f oll owi ng expl anati on: mi cr ot ubul es may sti mul at e acti vit y of kat ani n by f acilit ati ng kat ani n-kat ani n i nt eracti ons, because when concentrati on i s l ow it i s mor e li kel y t hat n-kat ani n mol ecul es will bi nd near one anot her on t he mi crot ubul e, but hi gh concentrati ons of mi cr ot ubul es may r educe ATPase and severi ng acti viti es by pr eventi ng kat ani n-kat ani n associ ati ons, because, it i s mor e li kely t hat n-kat ani n mol ecul es wi ll be
di spersed over a hi gh number of mi cr ot ubul es and wi ll have l ess chance t o bi nd near one anot her ( Hart man and Val e, 1999).
Fi g. 1. 5: Effect of mi crot ubul e concentrati on on kat ani n’s acti vit y. ( Hart man and
Val e, 1999)
Acti vit y of kat ani n mi ght be r egul at ed by it s synt hesi s and degr adati on l evel s. It was found t o be t he case i n axons wher e kat ani n l evels are hi gh duri ng t heir most acti ve phases of gr owt h but drop once axons have contact ed t heir t ar get s ( Karabay et al., 2004) Ho wever, kat ani n i s wi del y di stri but ed and t here shoul d be anot her cont r ol mechani s m t o severe microt ubul es onl y when i t i s needed. There were some factors (like cyclinB/cdc2, cyclinB/cdc1) discovered that changed microtubule-severing activity in M-phase Xenopus egg extract. However, experiments with isolated katanin showed that it is not directly activated or phosphorylated (McNally & Thomas, 1998; McNally et al. 2002). Activity of katanin might be regulated indirectly by other MAP’s which would restrict accession of katanin to microtubules (Baas & Qi ang, 2005).
As r egar ds its bi ol ogi cal r ol e, kat ani n i s supposed t o be i mport ant i n mit osi s. It was me nti oned above t hat mi t oti c spi ndl e consi sts of mi cr ot ubul es. Micr ot ubule severi ng acti vit y it self was first i dentifi ed and st udi ed i n mi t oti c extract s of Xenopus l eavi s ( Mc Nall y & Val e, 1993). Kat ani n was f ound t o be hi ghl y concentrat ed at centr oso mes t hr oughout t he cell cycl e ( Mc Nally et al., 1996). Thi s support s t he
hypot hesi s t hat kat ani n me di at es t he di sasse mbl y of mi cr ot ubul e mi nus ends duri ng pol e war d fl ux (see Fi g. 1. 6) ( Mc Nall y et al., 1996; Quar mby 2000; Bust er 2002).
Fi g. 1. 6: Model of kat ani n’s r ol e i n t he mit oti c spi ndl e. Severi ng of t he ki net ochor e
mi cr ot ubul e by kat ani n rel eases a s hort mi cr ot ubul e frag ment ( A- B) and all ows t he ki nesi n-li ke pr ot ei n t o pull t he ki net ochore mi cr ot ubul e t owar d t he centr oso mal mat ri x ( B). Si mult aneous pol y meri zati on of t ubuli n occurs at t he pl us end ( B- C). ( Mc Nall y et al., 1996)
It i s al so s upposed t hat kat ani n f acilitat es mi cr ot ubul e depol y meri zati on i n t r ansiti on from i nt er phase t o mit osis by r el easi ng centr oso mal mi cr ot ubul es and di scl osi ng t heir mi nus end. Rel ease of mi cr ot ubul es fr o m centroso me i s essenti al i n neur ons and it is especi all y acti ve i n neur ons co mpar ed t o nonneur onal cells as it was menti oned.
1. 2. 3. Kat ani n subuni ts
As it was pr evi ousl y menti oned, kat ani n consi sts of t wo s ubunits: p60 and p80 whi ch wer e na med accor di ng t o t heir mol ecul ar wei ght . It has been s ho wed t hat p60 has mi cr ot ubul e-sti mul at ed ATPase and mi cr ot ubul e severi ng acti viti es i n t he absence of p80, whil e p80 t ar get s katani n t o centr oso me. ( Hart man et al., 1998) Ho we ver, t hese fi ndi ngs t ur ned out t o be mor e co mpl ex aft er fut her i nvesti gati ons.
p80 s ubunit i s 658 a mi no aci d l ong (rat p80) and cont ai ns si x ‘ ‘ WD40’ ’ repeat motifs ( Kar abay et al., 2004). These proteins are characterized by the presence of repeats consisting of between 40 and 60 amino acids with two internal conserved dipeptide sequences, glycine--histidine (GH) and tryptophan--aspartic acid (WD) Despite their highly conserved structural motif, WD40 proteins play very diverse functions. This is probably due in part to the ability of these proteins to coordinate the binding of a variety of proteins through their individual blades (Smith et al., 1999). The C-t er mi nal r egi on of p80 does not exhi bit si gnifi cant a mi no aci d i dentit y t o any pr evi ousl y described pr ot ei n. Alt hough p60 severs mi cr ot ubul es i n t he absence of p80, p80 was f ound t o affect t hi s aciti vit y. p80 can enhance p60 medi at ed mi cr ot ubul e severi ng by i ncreasi ng affi nit y of p60 t o mi cr ot ubul es ( Mc Nall y et al., 2000). p60/ p80 was f ound t o have a t wo-fol d hi gher mi cr ot ubul e severi ng activit y co mpar ed t o p60 al one. On t he ot her hand, i t was al so found t hat WD40 domai n cont ai ned i n p80 act s as a negati ve regul at or of mi cr ot ubul e severi ng by p60. ( Mc Nall y, Thomas, 1998) The sa me do mai n i s responsi bl e f or p80 centroso me t ar geti ng ( Hartma n et al., 1998; Mc Nall y et al., 2000; Mc Nall y, Tho mas, 1998).
1 M A T P V V T K T A W K L Q E I V A H A S N V S S L V L G K A S G R L L A T G G D D C R V N L W S I N K P N C I M S L T 6 1 G H T S P V E S V R L N T P E E L I V A G S Q S G S I R V W D L E A A K I L R T L M G H K A N I C S L D F H P Y G E F V 1 2 1 A S G S Q D T N I K L W D I R R K G C V F R Y R G H S Q A V R C L R F S P D G K W L A S A A D D H T V K L W D L T A G K 1 8 1 M M S E F P G H T G P V N V V E F H P N E Y L L A S G S S D R T I R F W D L E K F Q V V S C I E G E P G P V R S V L F N 2 4 1 P D G C C L Y S G C Q D S L R V Y G W E P E R C F D V V L V N W G K V A D L A I C N D Q L I G V A F S Q S N V S S Y V V 3 0 1 D L T R V T R T G T V T Q D P V Q A N Q P L T Q Q T P N P G V S L R R I Y E R P S T T C S K P Q R V K H N S E S E R R S 3 6 1 P S S E D D R D E R E S R A E I Q N A E D Y N E I F Q P K N S I S R T P P R R S E P F P A P P E D D A A T V K E V S K P 4 2 1 S P A M D V Q L P Q L P V P N L E V P A R P S V M T S T P A P K G E P D I I P A T R N E P I G L K A S D F L P A V K V P
4 8 1 Q Q A E L V D E D A M S Q I R K G H D T M F V V L T S R H K N L D T V R A V W T T G D I K T S V D S A V A I N D L S V V 5 4 1 V D L L N I V N Q K A S L W K L D L C T T V L P Q I E K L L Q S K Y E S Y V Q T G C T S L K L I L Q R F L P L I T D I L 6 0 1 A A P P S V G V D I S R E E R L H K C R L C F K Q L K S I S G L V K S K S G L S G R H G S A F R E L H L L M A S L D
Fi g. 1. 7: Ratt us norvegicus p80- kat ani n a mi no aci d sequence ( Gene Ba nk, t he
Eur opean Mol ecul ar Bi ol ogy Labor at or y, accession No. Q8 BG40). WD mot ifs ar e sho wn i n bol d, conser ved di pepti de sequences are underli ned.
Rat p60 i s a 491 a mi no aci d l ong pol ypepti de (Kar abay, 2004), it has a conser ved 230- a mi no aci d ATPase do mai n (see Fi g. 1. 8). BLAST search r eveal ed t hat ort hol og of p60 exi sts i n C. el egans, i n Ar abi dopsi s t hali ana and t here are vert ebr at e ho mol ogs of p60 ( Hart man et al., 1998; St oppi n-Mel l et et al., 2003; Bouqui n et al., 2003, Yang et al., 2003). p60 al one di spl ays a mi cr ot ubul e-sti mul at ed ATPase acti vit y. Alt hough, when co mpari ng t he r at es of mi cr ot ubul e di sasse mbl y, p60 i s half as acti ve as p60/ p80 ( Hart man et al., 1998).
1 M S L L M I T E N V K L A R E Y A L L G N Y D S A M V Y Y Q G V L D Q I N K Y L Y S V K D T H L H Q K W Q Q V W Q E I N 6 1 V E A K H V K E I M K T L E S F K L D S T S L K A A Q H E L P S S E G E V W S L P V P V E R R P L P G P R K R Q S T Q H 1 2 1 S D P K P H S N R P G A V V R A H R P S A Q S L H S D R G K A V R S R E K K E Q S K G R E E K N K L P A A V T E P E A N 1 8 1 K F D S T G Y D K D L V E A L E R D I I S Q N P N V R W Y D I A D L V E A K K L L Q E A V V L P M W M P E F F K G I R R 2 4 1 P W K G V L M V G P P G T G K T L L A K A V A T E C K T T F F N V S S S T L T S K Y R G E S E K L V R L L F E M A R F Y 3 0 1 S P A T I F I D E I D S I C S R R G T S E E H E A S R R V K A E L L V Q M D G V G G A S E N D D P S K M V M V L A A T N 3 6 1 F P W D I D E A L R R R L E K R I Y I P L P S A K G R E E L L R I S L R E L E L A D D V N L A S I A E N M E G Y S G A D 4 2 1 I T N V C R D A S L M A M R R R I E G L T P E E I R N L S R E E M H M P T T M E D F E M A L K K V S K S V S A A D I E R 4 8 1 Y E K W I V E F G S C
Fi g. 1. 8: Ratt us norvegi cus p60- kat ani n a mi no aci d sequence ( Gene Ba nk, t he
Eur opean Mol ecul ar Bi ol ogy Labor at or y, accessi on No. AY621629). Part of p60 sho wn i n bol d i s t he sequence whi ch was cl oned and expr essed; underli ned part of p60 is t he conser ved C-t er mi nal AAA do mai n.
Kat ani n or its ort hol ogs ar e f ound i n a nu mber of or gani s ms: sea ur chi n, Xenopus
l aevi s, Chl amydo monas s p., Arabi dopsi s t hali ana, r at, hu man. Thi s micr ot ubul e
severi ng pr ot ei n has an i mport ant r ol e i n pr ocesses wher e mi cr ot ubul e r eorgani zati on is required, e. g. mit osi s, axonal gr owt h, defl agellati on, t heref ore it i s ext ensi vel y st udi ed. Full charact eri zati on of pr operti es and f uncti ons of bot h s ubunits of kat ani n woul d enabl e t o bett er underst and acti vit y and rol es of kat ani n itself.
Anti bodi es agai nst p80 and p60 wi ll be used i n t he st udi es on separat e subuni t s or on a whol e di mer pr ot ei n. For most r esearch, di agnosti c, and t herapeuti c pur poses, monocl onal anti bodi es, deri ved fr o m a si ngl e cl one and t hus s pecifi c f or a si ngl e epit ope, are pr eferabl e (Gol dsby et al., 2000). Reco mbi nant sol ubl e pepti de woul d pr ovi de best conditi ons for monocl onal anti body pr oducti on.
Bot h kat ani n s ubunits (p60 and p80) have been s ought t o be expr essed, si nce separati on of t he nati ve subunits requires denat urati ng conditi ons. Ho wever, bact eri al expr essi on of p60 pr oduced l ar gel y i nsol ubl e pr ot ei n ( Hart man et al. 1998). Over pr oducti on of het erol ogous pr ot ei ns i n t he cyt opl as m of E. coli i s oft en acco mpani ed by t heir mi sf ol di ng and segregati on i nt o i nsol ubl e aggr egat es kno wn as i ncl usi on bodi es ( Baneyx, 1999b). Due to solubility problems related to the bacterial expression of p60 subunit synthetic anti-peptide antibodies have been used until now.
Taki ng i nt o account t he need f or sol ubl e p60-kat ani n pepti de, we have set t he foll owi ng obj ecti ves for thi s research:
- t o cl one and expr ess p60-kat ani n;
- t o adj ust expressi on conditi ons i n or der t o achi eve sol ubl e p60- kat ani n; - t o purify pr oduced pr ot ein s o t hat it coul d be f urther used f or monocl onal
2. MATERI ALS AND METHODS 2. 1 Mat eri al s
2. 1. 1 Equi p me nt
Cent rifuge, Beck man coult er, Avanti J-30I
DNA sequencer, Appli ed Bi osci ences 3100- Avant Gel dr yer, Bi o- Rad 583
Mi ni centrifuge, Hetti ch zentrifugen EBA 21 Mi ni - Verti cal Gel Syst em, Bi o Worl d EC 120 Or bit al shaker, For ma
Pr eci si on wei gher, Preci sa 620 C SCS
Rocki ng shaker, Hei dol ph i nstru ment s Duo max 1030 Ther mo mi xer co mf ort, Eppendorf
UV spectrophot o met er, Beck man DU530 Life Science UV spectrophot o met er, Shi madzu 160
Vort ex Hei dol ph, Reax top
2. 1. 2 Che mi cal s and enzy mes
I PTG (isopr opyl- β- D- 1-thi ogal act opyranosi d) Appli Che m
Eco RI restri cti on enzy me
Mass Rul er ™ DNA Ladder ( Mix, 80bp- 10Kb)
Page Rul er ™ Prest ai ned Pr ot ei n Ladder (10- 170 kDa) Fer me nt as
Abs ol ut e et hanol Abs ol ut e met hanol Aceti c aci d (gl aci al) Ca Cl2
Gl ycer ol Isopr opanol KH2PO Na2HPO4 Na Cl NH4Cl
Phos phori c aci d Fl uka
T7 pri mer (5' d[ TAATACGACTCACTATAGGG] 3') I nvitrogen
Acr yl a mi de Br o mophenol bl ue EDTA
Et hi di u m br o mi de Gl ucose
2- mer capt o-et hanol Mg Cl2 Na H2PO4 Na OH SDS TE MED Tri s Yeast extract Me rck
Hi nd III restri cti on enzyme Ne w Engl and Bi ol abs
T4 DNA li gase
Loadi ng dye Pro mega
Ni - NTA agar ose for His-tag fused pr ot ei n purifi cati on Qi agen
Agar ose AMPS Gl yci ne Tet racycli n Si g ma 2. 1. 3 Buffers TAE Buffer (50X) 242 g Tri s base (40 mM)
57. 1 ml gl aci al aceti c acid ( 20 mM) 100 ml 0. 5 M EDTA ( pH 8. 0) (1mM) H2O up t o 1 liter
Lysi s Buffer for Met al Af fi nity Chro mat ography
34. 5 mg Na H2PO4 (50 mM) 87. 7 mg Na Cl (300 mM) 3. 4 mg i mi dazol e (10 mM)
H2O up t o 5 ml, adj ust pH t o 8. 0 usi ng Na OH
Was h Buffer for Met al Af fi nity Chro mat ography
69 mg Na H2PO4 (50 mM) 175. 4 mg Na Cl (300 mM) 13. 6 mg i mi dazol e (20 mM)
H2O up t o 10 ml, adj ust p H t o 8. 0 usi ng Na OH
El uti on Buffer for Met al Affi nity Chro mat ography
34. 5 mg Na H2PO4 (50 mM) 87. 7 mg Na Cl (300 mM) 2. 72 g i midazol e ( 4 M)
Buffers of restri cti on and li gati on enzy mes
Buffers of restri cti on and li gati on enzy mes wer e pur chased t oget her wit h t he enzy mes.
2. 1. 4 Speci al reagents and kits
Agar ose Gel DNA Extracti on Kit, Roche
Hi gh Pure Pl as mi d Isol ati on Kit f or s mall-scal e (mi ni ) preparati ons, Roche 50 % Ni- NTA agar ose suspensi on, Qi agen
Anti- His6 mouse monocl onal anti body, Roche Goat AP conj ugat e anti-mouse, Novagen
2. 1. 5 Bact eri al strai ns
Escheri chi a coli strai n XL1 Bl ue [recA1 endA1 gyrA96 thi-1 hsdR17
supE44relA1 lac [F' proAB lacI qZΔM15 Tn10 (Tet r)]], Novagen
Escheri chi a coli strai n BL21 ( DE3) pLys S F-- dc m o mpT hsdS(r B-- mB--) gal λ( DE3)[ pLys S Ca mr], Novagen
2. 1. 6 Bact eri al cult ure me di a
LB me di u m was pr epared by di ssol vi ng 10 gr a m trypt one, 5 gr a m yeast ext ract, and
10 gr a m Na Cl i n di stilled wat er. Di stilled wat er was added t o a fi nal vol u me of one liter. The LB me di u m was st erilized by aut ocl aving f or 15 mi nut es. I n or der t o ma ke sel ecti on medi a, anti bioti c was added t o t he LB me di u m accor ding t o t he concentrati on descri bed i n Tabl e 2. 1, and t he antibi oti c cont ai ni ng LB wa s st or ed at 4º C.
LB- agar pl ate was pr epared by addi ng 15 gra m/l of agar t o LB me di u m and
st erilized by aut ocl avi ng as descri bed above.
S OC me di u m was used t o culti vat e E. coli f or 1 hour aft er t e mper at ure shock duri ng
transf or mati on. It was prepared by di ssol vi ng 2 gr a m of t r ypt one, 5 gr am of yeast extract, 0. 058 gr a m of Na Cl, 0. 0186 gr a m of KCl , 0. 095 gr a m of Mg Cl2, 0. 24 gr a m of MgS O4, 0. 36 gr a m of gl ucose i n di stilled water. Di still ed wat er was added t o a fi nal vol u me of 100 ml. The SOC me di u m was st erili zed by aut ocl avi ng at 120° C f or 15 mi nut es. When r equi red, anti bi oti c was added t o t he medi u m i n or der t o make sel ecti on ( Tabl e 2. 1).
Tabl e 2. 1. St ock and worki ng sol uti on of anti bioti c Anti bi oti c St ock sol uti on
concent rati on
Wo r ki ng concent rati on
Ka na myci n 10 mg/ ml i n wat er 50 μg/ ml
Tet racycli n 15 mg/ ml i n et hanol 15 μg/ ml
2. 1. 7 T/ A cl oni ng vect or wit h cl oned i nsert
pCR 2. 1- TOP O (I nvitrogen) wit h cl oned f ull l engt h p60 s ubunit was pr evi ousl y desi gned by Dr. Ar zu Karabay. The cl oned p60 i s i n r everse directi on i. e. i t s codi ng strand and non- codi ng strands exchanged t heir positi ons: 5’ of codi ng strand of p60 i n nor mal directi on st arts wit h a st art codon ATG, whi ch i s at 5’ of non-codi ng strand of t he reversel y cl oned p60 (see Fi g. 2. 1)
2. 1. 8 Expressi on vect or
2. 1. 8. 1 pET Expressi on Syste m
In pET s yst e m ( pl as mi d of expr essi on by T7 pol ymer ase), t he expr essi on of t he DNA construct i s under t he contr ol of T7 pr o mot er, whi ch i s r ecogni zed by t he T7 DNA dependent RNA pol y merase (transcri pt ase), but not E. coli RNA pol y mer ase. Typi call y, T7 t ranscri pt ase i s expr essed by t he host geno me, whi ch i s i nsert ed behi nd t he l ac UV5 pr o mot er. The l att er i s i nsert ed i nt o geno me vi a l a mbda DE3 phage. Ther ef ore, t he host strai ns t hat carr y T7 pol ymer ase i n t heir geno me under t he contr ol of I PTG-i nducabl e l ac UV5 are call ed DE3.
Whi l e t hi s syst e m l eads t o t he s ynt hesi s of l ar ge a mount s of mRNA, and, i n most cases, t he conco mit ant accu mul ati on of t he desired pr ot ei n at ver y hi gh concentrati ons ( 40-- 50 % of t he t ot al cell pr ot ei n), it i s not wit hout dr a wbacks. For exa mpl e, hi gh l evel of mRNA can cause ri bosome destructi on and cell deat h, and leaky expr essi on of T7 RNA pol y mer ase may r esult i n pl as mi d or expr essi on i nst abilit y. Furt her more, even ‘e mpt y’ pET pl as mi ds are t oxi c t o E. coli i n t he pr esence of I PTG. So me of t he strat egi es t hat have been devel oped t o addr ess t hese issues are co- overexpressi on of phage T7 l ysozy me ( whi ch degr ades T7 RNA pol y mer ase) fro m t he compati bl e pLys S and pLys E pl as mi ds ( Novagen) and t he
Fi g. 2. 1: Vect or map of p CR2. 1- TOP O wi t h cl oned i nsert. Cl oni ng/restricti on ar ea
wi t h sche mati call y r epresent ed i nsert i s sho wn i n a l ar ger scal e. Arr ow stret chi ng from PCR pr oduct poi nt s at t he part of cl oned p60 s ubunit ( begi nni ng and end of t he sequence) i n a l ar ge scale. At t he bott om-l eft part of t he pi ct ure part of nor mall y ori ent ed p60 is sho wn i n itali c.
i nserti on of a l ac operator sequence do wnstrea m of pl as mi d-encoded T7 pr o mot ers, i n or der t o reduce l eaky transcri pti on ( Mierendorf et al., 1994; Baneyx 1999).
2. 1. 8. 2 pET- 30a vect or
The pET- 30a( +) ( Novagen) vect or was ki ndl y provi ded by Dr. St ephan Scheur er ( Paul- Ehrli ch-I nstit ut e, De pt. of All er gol ogy). The pET- 30a ( +) vect or carri es an N-ter mi nal Hi soTag ®/t hr ombi n/ SoTag ™/ ent eroki nase confi gurati on pl us an opti onal C-ter mi nal Hi soTag sequence. The circul ar map ( Fi g. 2. 2.) and t he cl oni ng/ expr essi on regi on ( Fi g. 2. 3.) are shown bel ow. The vect or is 5421 bp l ong.
5’ TCG. GCT. TGC. TTC ….. AAG. ACT. CAT3’ 3’ AGC. CGA. ACG. AAG….. TTC. TGA. GTA5’
Nor mall y ori ent ed p60
5’ ATG. AGT. CTT …AGC. CGA3’ 3’ TAC. TCA. GAA …TCG. GCT5’
Fi gure 2. 2: Vect or map of pET30a. Source: htt p:// www. e mdbi osci ences. com
Fi gure 2. 3: Cl oni ng/ expressi on regi on of t he codi ng strand of pET30a.
2. 2. METHODS
2. 2. 1 Small scal e pl as mid DNA preparati on ( mini - prep)
Pl as mi d mi ni pr eparati on was perf or med usi ng Roche, Hi gh Pur e Pl as mi d Isol ati on Ki t f or s mall-scal e ( mi ni) pr eparati ons, f oll owi ng i nstructi ons of t he ma nuf act urer. The pri nci pl e of t hi s purifi cati on i s as f oll ows: al kali ne l ysis r el eases pl asmi d DNA from bact eri a and RNase r e moves all t he RNA i n t he l ysat e. Then, i n t he presence of a chaotr opi c salt ( guani di ne HCl ), pl as mi d DNA bi nds sel ecti vel y t o gl ass fi ber fl eece i n a centrifuge t ube. The DNA r e mai ns bound whil e a seri es of r api d ‘ ‘ wash-and-spi n’ ’ st eps r e move cont a mi nati ng bact eri al component s. Fi nall y, l ow s alt el uti on re moves t he DNA fr o m the gl ass fi ber fl eece.
The pr ot ocol is as foll ows:
A si ngl e bact eri al col ony was pi cked and i noculat ed i nt o 10 ml LB me di a ( wit h kana myci n) cont aini ng Fal con t ube, and gro wn over ni ght wit h vi gor ous shaki ng (250 rpm) at 37ºC.
The f oll owi ng day, 7. 5 ml of t he cult ure was distri but ed i nt o 5 eppendorf t ubes ( 1, 5 ml each t ube), and t he bact eri a wer e recovered by cent rifugation for 5 mi nut es at 14, 000xg. The super nat ant s wer e discar ded.
The bact eri al pell et was r esuspended i n 50 μl of suspensi on buffer i n each eppendorf t ube separat ely and t hen coll ect ed t o one eppendorf t ube ( 250 μl suspensi on buffer i n t ot al). Suspensi on buffer cont ai ns RNase whi ch r e moves bact eri al RNA.
To l yse t he cells, 250 μl l ysis buffer was added (cont ai ns Na OH), mi xed by i nverti ng t he t ube 6 ti mes and i ncubat ed at r oom t e mper at ure f or up t o 5 mi nut es.
Lysis was st opped by additi on of 350 μl i ce-cold bi ndi ng buffer. Tube was agai n i nvert ed 6 ti mes and i ncubat ed on i ce for up t o 5 mi nut es.
The mi xt ure was centrifuged f or 10 mi nut es at 14. 000 x g and t he super natant was t ransferred t o a filter t ube. Chr o mos o mal DNA was pr eci pit at ed wi t h cell ul ar debri s duri ng centrifugati on and t hi s supernat ant cont ai ns t he pl as mi d DNA.
Agai n centrifugati on f or 1 mi nut e at maxi mum s peed was perf or med. Pl as mi d DNA i s bound t o t he gl ass fi bers pr e-packed i n t he filt er t ube. Super nat ant was di scar ded fro m t he coll ecti on t ube.
To wash t he cells, 700 μl of wash buffer was added t o t he filt er t ube and centrifuged at maxi mu m speed f or 1 mi nut e. Supernat ant fr o m coll ecti on t ube was di scar ded.
To el ut e t he DNA 100 μl el uti on buffer was added, and t he DNA s ol ution was obt ai ned by centrifugati on for 1 mi nut e at full speed.
2. 2. 2 Deter mi nati on of nucl ei c aci d concent ration
Recover y, purit y and concentrati on of nucl ei c aci ds wer e det er mi ned by spectrophot o met ri c analysi s. The r ati o of absorbance at ( A260) shoul d be 1. 8 f or DNA. I n t he pr esence of pr ot ei n cont a mi nati on, t he r ati o i s l ess. For t he meas ure ment, we used a s pectrophot o met er fr o m Shi madzu. The DNA was dil ut ed 1: 200 or 1: 2000 i n di stilled wat er and t ransferred t o a quart z cuvett e. The abs or pti on was at wavel engt h of 260 n m. An opti cal densit y (OD) of 260 n m of 1. 0 i s equi val ent t o 50 μg/ ml DNA. The f or mul a used t o cal cul at e t he concentrati on ( C) i s t he foll owi ng:
C = OD 260n m x dil uti on factor x equi val ent = x μg/ ml
In or der t o check r esults of t he s pectrophot o met ric anal ysis, DNA ext ract ed fr o m gel was al so run on t he 1 % agar ose gel bef ore li gati on.
2. 2. 3 Preparati on of chemi call y co mpet ent cells -- cal ci um chl ori de method
To i ntr oduce pl as mi d DNA or r eco mbi nant pl asmi d DNA i nt o bact eri a, t he bact eri a had t o be made co mpet ent f or t hi s pur pose. We home ma de co mpet ent cells accor di ng t o t he foll owi ng pr ot ocol, pr ovi ded by Sa mbr ook et al ( Sa mbr ook et al., 1989).
Wor ki ng asepti call y, XL1 Bl ue cells (t aken fr om a gl ycer ol st ock cultur e) wer e streaked out on an LB pl at e and i ncubat ed over ni ght at 37° C.
The next day, one bact erial col ony was pi cked and i nocul at ed i nt o 10 ml of LB me di u m cont ai ni ng t etracycli n i n a Fal con t ube, and t he over ni ght cul t ure was gr own.
The next day 100 ml LB me di u m was i nocul ated wit h 4 ml of over night cult ure sol uti on and was i ncubat ed at 37° C i n a r ot at or y s haker. Cell densit y was measured by a s pectrophot o met er at OD6 00. Whe n an OD6 00 of 0. 6 was reached, t he bact eri a wer e t ransferred t o 50 ml pr echill ed st eril e ultracentrifuge t ubes and i ncubat ed on i ce for 10 mi n.
The cells wer e spun do wn at 1600 x g f or 10 mi nut es at 4º C, t he super natant was di scar ded
Bact eri al pell et was r esuspended i n 10 ml i ce-cold Ca Cl2 ( pH 7, t he s ol ution was filter st erilized t hr ough a filter of 0. 45 μ m pore si ze) and i ncubat ed on i ce for 30 mi nut es.
Centrifugati on was perfor med agai n f or 5 mi nut es at t he sa me s peed as pr evi ousl y, and t he cells wer e resuspended i n 2 ml of Ca Cl2.
The cells wer e used i mme di at el y f or transf or mati on and/ or di stri but ed i nt o pr echill ed st eril e mi cr of uge t ubes. The co mpet ent cells wer e st ored at -- 80º C i n 40 μl ali quots.
Ca Cl2 sol uti on
Cont ents Concent rati on Amo unt
Ca Cl2 60 mM 0. 33 g
PI PES 10 mM 0. 15 g
Gl ycer ol 15 % 7. 5 ml
wat er X up t o 50 ml
2. 2. 4 Transf or mati on of co mpet ent cells
Tr ansf or mati on i s t he t er m used t o descri be t he i ntroducti on of pl as mi d DNA i nt o bact eri a. The experi ment can be perf or med by an el ectri cal or a che mi cal met hod. We used t he che mi cal met hod whi ch consi sts of a heat shock t o i ntr oduce t he DNA i nt o t he host. A short pr ot ocol is as foll ows:
The co mpet ent cells fr om -- 80o C wer e t ha wed on i ce. 2- 3 μl of purifi ed pl as mi d DNA or 10 μl of li gati on mi xt ure was added t o 20 μl of co mpet ent
cells and t he eppendorf t ube cont ai ni ng t he cells was i ncubat ed on i ce f or 30 mi nut es.
Aft er t hat, t he cells were kept at 42o C f or exactl y 40 seconds ( heat s hock) and i mmedi at el y wer e i ncubat ed on i ce for 10 minut es.
80 μl of LB li qui d medi u m was added t o co mpetent cells and t he eppendorf t ube was vi gor ousl y shaked at 37o C for 1 hour.
The cells wer e t hen pl ated ont o LB ( cont ai ni ng appr opri at e anti bi oti c) pl at e. The pl at es wer e i ncubat ed at 37o C over ni ght.
2. 2. 5 DNA cl eavage with restri cti on endonucl eases
Restri cti on enzy mes, also call ed r estri cti on endonucl eases are bact erial pr ot ei ns whi ch wor k as an i mmune s yst e m i n bact eri a. Their r ol e i s t o destroy bact eri ophages or ot her vir uses whi ch i nvade bact eri a. Restricti on endonucl eases r ecogni ze a specifi c nucl eoti de sequence, and cut DNA wher ever t hi s specifi c sequence i s f ound. Us uall y t he pali ndr o mi c r estri cti on sit es have a l engt h of 4 t o 8 base pairs. The purifi ed r estri cti on endonucl eases are co mmer ci all y avail abl e, and are used t o generat e DNA fr ag ment s f or cl oni ng experi ment s. Ther ef ore, r estri cti on endonucl eases are a maj or t ool i n reco mbi nant DNA t echnol ogy.
Si nce t he cl oned p60 had t o be s ubcl oned changi ng it s ori ent ati on at t he sa me ti me, t he foll owi ng require ments were set for t he restri cti on endonucl eases:
- t hey have t o cut bot h T/ A and expr essi on vect ors;
- t heir sequence i n t he vect ors shoul d be di fferent, i. e. 5’ r estri cti on sit e on T/ A vect or shoul d be i n 3’ co mpar ed t o anot her r estri cti on sit e on expr essi on vect or;
- restri cti on endonucl eases shoul d l eave out TGA codone i n non- codi ng strand of T/ A vect or;
- restri cti on endonucl eases shoul d not cut i nsi de t he p60 s ubunit, pr eferabl y.
Eco RI and Hi nd III met most of t he require ment s
Nor mall y DNA was cl eaved at 37º C f or 4 hours wit h 1 X buffer (supplied by t he ma nuf act urer).
Restri citon reacti on mixt ures
Cont ents Amo unt Vol u me, μl
Pl as mi d DNA ( pET 30a) 1. 5 μg 11, 5
EcoRI 10 units 1
Hi nd III 20 units 1
10 X Y buffer 1x 1, 5
Tot al reacti on vol u me 15
Pl as mi d DNA ( pCR2. 1- TOP O -p60) 4. 55 μg 7
EcoRI 10 units 1
Hi nd III 20 units 1
10 X Y buffer 1x 1
Tot al reacti on vol u me 10
2. 2. 6 Li gati on
T4 DNA l i gase i s encoded by t he gene 30 of bacteri ophage T4. Thi s enzyme can be used t o li gat e DNA r estricti on frag ment s. T4 DNA l i gase has t he capacit y t o cat al yze
i n vitro t he f or mati on of a phosphodi est er bond bet ween adj acent nucl eoti des, one
cont ai ni ng a t er mi nal 5-phosphat e gr oup and one cont ai ni ng t he hydr oxyl t er mi nus. A r evi e w of t he li gati on reacti on can be seen i n Cher epanov and de Vri es, 2003. I n t his st udy, cohesi ve li gation t ype was perf or med.
Most r estri cti on endonucl eases creat e cohesi ve ends. When frag ment DNA a nd pl as mi d DNA ar e di gested wi t h t he sa me r estri cti on endonucl ease, co mple ment ar y cohesi ve ends ar e generat ed t hat can easil y be li gat ed. For li gati on over ni ght i ncubati on at r oo m t e mperat ure was perf or med. Li gati on mi xt ur e was ma de by addi ng co mpounds depi cted bel ow:
Cont ents Amo unt Vol u me, μl
Pl as mi d DNA 300 ng 1
Insert DNA - 7
10 x buffer 1x 1
Li gase 1
2. 2. 7 Agarose gel el ectrophoresi s
Agar ose i s a li near pol ymer co mposed of r esi dues of D- and L- gal act ose and i s used t o separat e frag ment s of DNA or RNA by si ze ( Sa mbr ook and Russell, 1989). Si nce deoxyri bonucl ei c aci ds are negati vel y char ged, t hey mi grat e t hr ough t he agar ose gel i n a el ectri cal fi el d t owards t he positi ve anode. Mol ecul es of doubl e-stranded DNA mi gr at e t hr ough gel mat ri ces at r at es t hat are i nversel y pr oporti onal t o t he l og10 of t he nu mber of base pairs, t heref ore s mall mol ecul es mi gr at e f ast er t han large ones. Pl as mi d DNA or DNA f rag ment s obt ai ned aft er treat ment wit h r estri cti on enzy mes wer e separat ed by el ectrophor esi s t hr ough a l ow melti ng poi nt agar ose gel. 1 % agar ose concentrati on was used.
To prepare 1 % agar ose gel, 0. 4 g of l ow melti ng poi nt:
Agar ose was di ssol ved i n 40 ml (s mall gel) 1x TAE ( Tris-acet at e- EDTA) buffer.
The agar ose was sol ubilized i n a mi cr owave oven until t he agar ose was co mpl et el y di ssol ved.
Gel was cool ed t o ≤ 45º C and et hi di u m br o mi de was added t o a fi nal concentrati on of 0. 5 μg/ml and mi xed t hrough gentl e s wirli ng.
The agar ose gel was t hen pour ed i nt o a hori zontal gel tray, and a co mb f or for mi ng t he sa mpl e sl ot s was pl aced i nt o t he gel.
The gel was s oli difi ed f or about 30 mi nut es and t hen pl aced i nt o an el ectrophor esi s t ank, where t he gel was cover ed by 1x TAE buffer used t o ma ke t he gel.
The DNA was mi xed wi th l oadi ng dye and t he sampl e was pl aced i nt o a well on t he agar ose gel. As frag ment si ze contr ol, a MassRul er ™ DNA Ladder, Mi x ( 80bp-10kb) was used. El ectrophor eti c separati on was achi eved by const ant current at 80 mV f or 60 mi nut es.
DNA wi t hi n agar ose gel s i s onl y vi si bl e when st ained wi t h et hi di u m br o mi de and can t hen be vi suali zed under UV l i ght. The gel was pl aced ont o an UV i l u mu mi nat or t hat e mit s UV l i ght at 302 nm and phot ographed wi th a ca mer a connect ed t o a co mput er. I mage fil es wer e saved wi t h UVI Phot o MW Ver si on 99. 05 f or Windo ws 95 & 98, UVIt ec Lt d. and subsequentl y anal yzed. The si ze of t he DNA was det ermi ned by co mpari ng t heir mobilit y wit h t he frag ment s of t he Mass Rul er.
