Histone analysis in Volvox

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VOL~OX

-- ~ I S T O N E A N A L Y S ] [ ~ g ] N

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DermisM.BI~ADLEY*::::-.

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H e r b e r t Ha ' G O L D I N "

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~ ~ @ o ? ~ e n $ ,of ~OllOgy. Tl~e ,L~i~.ezs~ty,of go~edo. Toledo. ,Ohio :43606. ;D~.4.

.

"

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and -

.

.

.

.

-

J Russell CLAYBROOK

-

.

-

.Dean. "1~e.n:?~gf . - P ~ s ~ o l o x y , ~ t e d i c ~ . 2 C o l l e g e o f O h i o ~ z T o l e d . O. T ~ l e ~ o , , G h i o 4 3 6 . 7 4 ,

USA

Recei,ed 4 ~a~ch ] 974

~.

qgan,~J!afive.diff.e~ences w e r e s e e n ~ e t w e e n ~ o m e of ~]e histone bands of the ~wo ,o~gm'~sms.

I n t r o d n ~ n

.HJs,ton.ez a r c ba~J.c prote,~ns as.socia~.ed w,~lh :the chr,0rrao~ornal co.mpl~:*, a n d a r e ~ h o u g h t t o p.'tay sorn~ r o l e i n g e n e .action ,at :thele'e.d o f ce,'llutar .diffexent~afi.on ~I]. V o i d e r , wi-% ~ts ,~w:o d~slVm,etee]t l~pe's; s o m a t i c :and r e p r o d u c t i v e , r,epresents -eellu]ar differ,en't~a'tion a t t h e sLm.~lest level. NuCleic ~ac~fls }~a,ee b e e n is.elated f r o m thi~; ~o_~gangsm I2], b,ut-we k n o w of n o inve~t~.gations d~alin;g w~th t]~e d e m o n ~ r a t i o ~ ,oftfist.one~ in Yo~ox. }Ve"now a - e p 0 r ~ ~ l ~ p r o t e i n s h~ve b e e n ~sola,,ed - f r , o m V o ! P o x . c a r r e r i w h i c ~ axe h~s~oneS b y - t h e f,'o]lowing c r i t e r i a : px.epazafive b et~a.vior, a m i n o a ¢iu prof'fle, a b s e n c e o f tryp~op.ha~a, ,~ltraviOlel a b s o r p l i o n , a n d d e e t r : o p h o r e ~ e pa%.ter~ A ~ t h o u g h n o a p p a r e n t v a r i a d 6 n i n : e t e e t r o p h 0 x ¢ f i = palt,ern w a s f o ~ _ d in " . ~hree d e v e l o p m e n t a l ~ a g . e s o f t h e !ife.-cyele, s o m ~ . quanTti~t~afive d ~ f e r e n c e s w e r e n o t e f l , - t n c~mpaa-ing ~t}le'~~dn~rS , o f c a i f 'th~m.us a n d :K. ~ r t e r . g , ~ i a ~ .e]eCt~opl~olefic_patt.erns w=re o b z e ~ . e d ; : i n a d d i t i o n ,

!:i !;

~

* This w0ik:Was~performed iu~prddal",/'ulfillmenI.of ihe " _ r~qu~qm..en~ts re! a Maste L of Scaence degree,.:

~'~ ':-~:;.,:;i.:. i :-: i; -:: ;:. -:: .::::. ::i : ; - i , ? : :

.....-:.

2. Methads T h e o r g a n i s m u s e d i n , h i s ~tt~dy w a s V o l ~ o x ~: ~ ' m g a r i e n s i a !y,e n g a r , staraL,a ~ - 10 ~_-,] A x e n i c c~tl..~.. ,e~ w e r e g r o w n i n m e . ~ m d e v i s e d b y Pr.o~'asoti and:Pin'tnex. A d . e t ~ l e d .descript~0n ,of fi~.e ,cu!ture ~e,ehniques a n d :the ,tife;cye!e WaS ~pr.esentedcarzeri

by Start

14]:,

-

.

'

T h e }aSs~one c.o~tenI -~,as d e ' t e r r a , n e d a t t h r e e ~ a g e s ; t ) ~ n r n e d i z t e l y af~ter 'the release .of yo'~ng ~ p h e m i d s , 2 ) b e f o r e cleavage O f ~he gonJd~a; a n d .: - 3 ) be.ore release ,-sfy,o~ang ~phero~dz. Samp)e~ o f " . e a c h ziage ~,,~ere C~itected .on f i l l e t p a p e r arid t h e w e t w e i g h t d e t e r m i n e d . A11 s u b s e q u e n t , st~ps w e r e c a n n e d o u t a~.0 ---4 Ca . •. •

.

,

T~e mSi6ne-ex~mcuon

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pro~u~e

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: , v ~ s a! m 0 d i '

eytbplasn-dc,.~.iape~natant~and

irfit]at a nuclear-rich p e l l e t , T h i s pel]e~ .was _ e s U s p e n u ~ d m t h e . g n n d t n g : -_

i ; ~.

,;)i /~::: ;~_:ii-)-.:17~:- i / - - ~:-~"ii .(; ~' , -i.;:

-:~V:~ium~.;:l)ii~rhbe;2 -:]

..................

t--

[5] ~Afier

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~ . . . . . . . . . . . . .

ele:ctrophoresis,

J

. . . . . . . .

t h e ge,]s ' w e r e

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y

'stained wlth

AmidoB]ack, and the ab:mrpfion p r o f i l e s , o f t h e .geLs - w e r e ~.:ob~[ained ~ 5 t h a P J i o t o v O l t . d e n s i t : o m e t e ~ " :(420:mp). :~ " _ : _ : • /.

of ~ , " y p t o p h a n

-The presence

was ±ested fo~ ina

por,fion :of £~e .mamp]e by ,the Hopk~s'Co]e ..

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-

The

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ultra'violet

from

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were

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a Sphaco

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10~7 5.9 s.4

!2.2 5-9 ~.~s

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0.O

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2.3

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O.O

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Basic]acidic

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LYS]ARG

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1.9 1.75

.1-:0 t.~67 "

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historic

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acids;

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crassa

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w o • :• - ", 2 6 0 . • " . a S O • " '300-" .: . • • • " ~ - ' " - , • --- ~ . - .... = . "-i yV:aVe.'"engtt#~Y ..:-.:-(:..: .~_:"..:?." '. {lable:2):. Thc ammo..ac~d :pro]~le:of .:the..K-ca~t~r . Fi.. ~ : - ; , , , _,-sL-_L:.: -: ~_:u_ " A}:~".; . , , .,,[ .... ' :,:=. i::.. " - - . , :.-:) ( " ': • - s a m p ] e s w a s . s ~ i t a r . k o tim t of]v: t;ms~:"The.-:comi=, ~ £ 1 J . ¥ : ~ o

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of/he -

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boW.me.seru._.m-a]bumin~ta studred.

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Ihe color xeacti,on,

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obseryedi~2

histcme

.,.Stag L ~ t ~ y p ! p p h a n a n d

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eonelud,ed

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the ~¢Ja~tivityof

mstones.prevmusly

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in the ,calf thymus.

~art:erior

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tha,t peaks "

0 00~ _~gtryptophan]~ng

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,color reaction

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a low, flatten:ed

m ~ z ' [ 7 ] L ..-..

No.ltryptophan

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22@--230

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0.5 1.23

: a n f i n o a c i d ~ e s o v e r e d . N o . c o r r e c t i o n s ~x,e r e a p p ~ e d f o r ~hy,dro]yfie to.'~es p f ; a m y ef-l~h.e a..:mino a d d s ~n ~z. -,-,-,-,-,-,-,-,-,-~TZt~2"£B a s S e ~n-D~.-a z e ~ d ~ ~ ; ] y a i n e ; a T . : ~ S n e az~d h i s i i d i n e ; a c i d o n e x az,e asp,at,tic a ~ d

extra,cte,d

.calf'thymus

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of standm'd

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w]-d,ch ~bSolbS sit~iongly m the

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13A " 5_3 7.7

3~1 7.6

" A ~ n o u n , S o f a m i n o a:cid nine expxesse~fl a s rn.oles per 10[} m o l e s , o f

" " .

spectra,(,fig,

to thespectrum

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I ~ . c a r Z e r i w,e~,e f o n r ~ d I:o b e e : s s e n f i a l ] y ~ d e a a t i c a ]

(3~gma),

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t ] .1 K3 .7.6 '

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Absorp~ibn from

9.7. " . " 8.2 " " " .4.'9 _ 5 . 9 " , s.s 5.7 "

. 'cysline]2

-

S. ,:]Resttlts-and discussion"

•

c ysteLne was anzlyz.ed.as

"

.:Glu~tamie Scribe . r ~ , , ~

M:et'hi,onine

analy.sis. -.After.

•

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acid oxidation,

acid..

::2.8 8.3

]~ol~ne Glyvine Phe~ay]alanine Tyros~e •

M o d e l .. ] 2 , D C m a i o m a t i c

,up fox..si:ngle column

( - : " " / : : : : . . / _ - - . _ i.". ." M a y 1 9 7 4

~sfidine

.. Iso].enc~ne.

: 300 mp .to 200 ~ . o n .a ' C ~ r y 1 4 r e c m d i n g speelrophotometer using a } mg:Jrfl calf thymus .Sistone . " • : s a m p l e . : ( S i g m a ) :fox c o m p a r i s o n . Amino acid profiles

" ::

A.<~,~ic

-Aianine "Valine Leuchae

method.

.

abs0ip;tio:r~ was determined

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..;.,-.-, . .

anti ;ranoz oi: .,v,:.carter~ an.a2¥:,

. ~ - t o [ n & .("Si~'~.a)"" "0'i" ]:: ~ } m l " ~-'i•:::"--":: ;-... ~"':." { . : .!?.[ ? ::i-..:.£]:qlo.'se]. ¢ s ~ i a n " d . ~

aifferea:ffom

:

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erassa

those: ofi;c~-.

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were-.

.:

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.

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:-.;-~/-: i, :.:-- _

-volume 4 1 o n U m b e / 2 '

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,F.'~.BS~ T T E R S -

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~ r a e i r g s o f ,~he : p M y a c r y l a , m i d e g e l s

shown in fig. t , M~grat~on is from r~gh! ~o left..A = ~tandazd calf. ~thym~ M~t.one; B = Y; ~ar~'en" ]~fis.~.one.Explana,lion m text. The bands p ~ i n g ~ are wariable in intengty and are believed :g'9~ep~.e~.en~fn-on-his.t.one~:on~-amirmn.~s. F~g. 2. Elec~-~pla0~efie pall.erns oflAstones f.lO~ calf ~thymu~ .(:geta) .anti V. ¢~zteri ~gv] b). Mig;afi~n i~ fxona top {+) :to boat.am ( - ) .

His~ones h~ve been ~,~,udiedex~ensively ~ ~gher organisms, p~tic~!arly ~ thymu~ and peab~d~,. and.. a p p e a l xo h a v e a r o l e in ~h.e
-

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. . . . . _ -~__ 7~-:~:~:~.~7.: v-~,~:4-;-~

-. -. . -:.---.:----:. :-::~ :.i , . . . .~t:.-: . . . . . . . . . . '~;se_br . ~~.mc~i~d:sia~::~a~v~i,~.. . .. . . . . . . . .-. . . . . . . . . . . :.__ "4c"-.:,'<':"[,41~:'Stah::-]R/.C.:(1969)k~rdts;-:~.bt~enk ~d 1 ! 1 , 1 ~ : 2 8 4 : , : - - - :"- - . :' . ; i .

~iic..o~ _ ga" . n ~

.ry.,~ -5-:2: ~ - : i ] ~ e ~ : : : ~ ] o ~ . ~ J s . L , - : 2 : 2 - . - - ,

.-. . . . .

.,xp

:-sl~cn:as-.~Oi.yox..vn'ay,p~r;,rm/~.-,t,! : . . _ _:_ ; . . .:~ _ - u . . . . . . . . .

:'::=:

.uU~o~-Or-."

:

¸,

-.2,. :;: -- -: :/.~ -.::!-.,. . . . ~v_'.:,:.7 ( ,- .: • . - - - : , ~ - ' ,: . . . . ~ :.",:-'- : ~ . ---.,. ~ -~onner J -el:at (1968) an:.z~letlloasan.t~nzym~l.-

.

:~enera~)oles-of~t0~aesbSi~elxt~;)~l~ S~i~,:~-:~; ":':,-i!:>;/::!::.-~.-":-:'(~ !. (g~-i~i!)

.

~"a~i"~i:0~¢; i¢ !;eas")~~o]- ~; i~ ~:i :appma-Ches:"~a ~ay :als0 reveal: im.vel)~)e.Cis .eft.": .::".:-:.., ";"-b"i;,.:'..:-~a~ic~s ::.N~.vo~i,il ".:"'.:.; ~""):-: ':'-:'.-],%~-C!:--:" .;. /:h~t0fie:ftmciions inchr;~moso~neiStrUcirkxe:::,andq~: ,-.-~; :]"..-!/"l:~] ::iieiarita;:R;,tewls;~; iaha w~mi,:D..: O962i!N.aiure-/

i~,cl~owledgements . " ..-: ; : -.. . . f , ) . : . - ) . "....:-- : . : ' . - -

..

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] :..; :.. :---/: . . . . - ~ 2 " : ::;:; _I.B]:B~fl~r:.L A.W,:Jqhns, Eo-:an¢i.~.~:~ tiI~1~i;I). M . e . . : :. : : " :-- ~/. • .... • .. " !iO96B)in;.:Pxbgr:::BiophYs_Mi~l..:BiO1. (Bu~tlex,:J:A:~..: -.

: ) - - :. W- ' ~ i. , b'"a n ' k: D . .~-R i c " ' h a. r ) ~ . -l C ."S , . o4 x ~ : , .' ~, ~Ohd

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10~ .. . .2. 0. 9 ; ~ e x~m.~ o•n ~ . ~. : e. s s. , ; ] q e• .w. .. .¥ ~ o ~. k ;

~(:1965%]-~Ones

a~a¢l Ot~e~

N~c,]eaz

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}'zoI,e~r~,

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laboral:ory and -for-.denac,ns~rahng ~he :sy.nchromza~ion.-. . _ -:.-~ .... -.--:.;-. : , : . ~ .... ' - a - .. • .v - : .... " " -'- - - . . . . - . : : - " ~;:: . : " - .i~, ' .....- -.... .pp;z.9--lgO=Aeaaem~c~ess,~e~'r,org; .. ..'. p r . O c e d u r e _ ~ , e . ~ s o , ~ h a n ~ D n J . ' R : o b e r t : ~ u n . a n o . t : o ~ . . : ,~ • -.. :,,_:-4, k . , ¢i~,=~,x ~ . x : ~ 6 _ Nia~.l Ae-'~ . , , & ~ , ~ . ." . . : . :. " . . ., , ,. , .. ~. • .... . .: , . , . . '. - . . . . : , ' = : " . nl.EtlJ~,-d:~ J..,. t . t y u J A ' ~ J ! " ~r.t. ~ - " t ' x-}. l u ~ ..~ .~ .- .2 Y i u J . . . . .,thei:use:of his .labo~atory-£acilitJes, San dra Swartz f o r .:. '...._ .: .-: N o ] , - , ( D a v i d s o n , : J . N : ~ n a C 0 f i m ; : W . !E.~ e d s . ) 1~- 2 5 ~ .. ::;~echnical-as.~sia-nCe .~'DrJ2ubi~HniliCa ~.~.: - . ~.. : . -

--

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~n tl~elamino'aCid for ..

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,ana]yses;

and::

his" c6mments ~Lon c e ~ n i n g -.. , • ....... : , . ".

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Atm.Rev.:g:ene!.

Sa:mi.ng; N.

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Bi.oc]aim:-Biophys.:

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S~:aan¢t

l=; n n c l S ~ e d ~ a n ; . ] ~ ;

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~Webb..N.W.C..

(i95~)

HiSf~hesa]na

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NS~,a~e ] 6'6; 7 8 6 ~ . N~c,~e05is~ones

"

iJ.t3] Far~brofigk,:D:;F~jin~am,F2~ndiBoniie~J-i(1968).. --. " '

"

Sleflmml,

P.ertman;C.

J_: t3]o~hem.

] { 1:~1] g e : s s o r a s ; .

N.

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57;5f-i?.:--

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:a~.d ~I~sk~;

2 • tl;

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