Fractionation of chick embryo cell RNA on columns of poly-l -lysine coated kieselguhr

4NALYTICAL

36, 278-287

BIOCHEMISTRY

Fractionation

of of

Chick

(1970)

Embryo

Poly-L-lysine

Coated

P. A. BROMLEY Vinls

L.aborato,y,

Cell

.4x11

December

on Columns

Kieselguhr

R. II. BARRY

Department of Pathology, Canzbtidge, England Received

RNA

Teks

Court

Road,

3, 19fj9

The principal methods available at present for the separation and characterization of RNA are sucrose density gradient centrifugat,ion (1)) polyacrylamide gel electrophoresis (2)) and chromatography on columns of met,hylatcd albumin coated kieselguhr (MAK) (3-5). Sucrose density gradient centrifugat,ion provides a means of separating various types of RNA on a preparat,ive scale, but the resolution of the method is poor. Polyacrylamide gel electrophoresis, on the other hand, provides a very high degree of resolution, but does not lend it,self readily to the fractionation of RNA in bulk. 129AK columns, although providing bulk separation of RNA, are difllcult to prepare and handle, and suffer from a lack of resolving power and reliability as well as a rather low capacity for RNA. Columns of polp-L-lysine coated kieselguhr (PLK) have been used for the fractionation of DNA from Bncilhs subtilis (6) and from Escherichia coli (7). This report describes the application of PLK chromatography t,o the fractionation of RiXA from primary cultures of chick embryo cells. Further characterization of the RNA peaks that eluted from the column was carried out by sucrose density gradient centrifugation. PLK chromatography scparat,es cellular RNA wit.21 a definition comparab’le to that obtained by polyacrylamide gel electrophoresis, and has the advantage of providing separation of RNA on a preparative scale. METHODS

“H-Uridine, s],ecific activity 27.8 Ci/mmole, Radiochemical Centre, Amcrsham, Bucks, England. Kicselguhr, Hyflo Superccl, Hopkin ‘9: Williams Ltd., Chadwell Heath, Essex, England. Poly-L-lysine HBr, minimum molecular weight 50,000, Koch-Light Laboratories, Colnbrook, Bucks, England. 278

FRACTIONATION

OF

RNA

ON

PLK

279

Culture medium 199 and calf strum, Burroughs Wellcome & Co., Ltd., London, England. Phosphate

BufJeretl

Strlirte

(Pl3S)

All PBS solutions containctl 0.02 dl KH,PO, and wvrc adjusted to pH sodium hydroxitlc. For gradient elution of the column, PBS containing 0.4 31 NaCl was :~lded to the 1ow-c~chamber, and PBS containing 4.0 31 NaCl was a(Idtvl to the upper chamber of the mixing device. For the stepwise clution 1>rocclclurc,inttvncdi:~tc PBS solut,ions cont,aining 0.7, 1.O, 1.3, 1.6, 1.9, anal 2.2 :I1 NaCl woe urt~l. 6.8 nith

Elcvvn (II\)- oltl chirk finbryos were clccapit,atr(l end cvisct~rated and the tissues minrc~tl mcll and stirred in 0.25% trypsin at 37°C for 1 hr. The cells n-erc filtcrcld through muslin and collected by low-speed cent*rifugation and rcs~~spc~~lcd in culture medium. The cells were seeded at a concentration of 0.5-1.0 X 10” cells ‘ml, I.50 ml being ntldcd to 2.5 liter glass roller bott’les, ant1 tliv cell:: groxn in 199 medium sul~plcmentetl with ‘105%calf s(‘rum, nt 37” for 48 hr. while rotating thv bottles at a ratcb of 0.6 rpm.

Isotopically labeled RX-4 was prepared hp incubating the cell cultures for 18 hr at 37°C in mcvlium containing 3 ,.,,Ci of W-uridine per ml. After washing, the ~11s n-crcl rcmorccl from the glass in :k sm:ill volume of 0.9% saline by gentle agitation with glas> beads, and were collected l)y low-speccl ccntrifugation. Thc~ RSA w:ts estrnctccl by gentle homogenizatimonof the cells with freshly rc~liatillcd watcbr snturntc(l phenol and nn equal volume of 2% sotlium tloclccyl sulfate, followed by vigorous shaking at 4” for 1 hr. The phase:: were .sqxrntccl by ccntrifugation at 2OOOqfor 10 min, and the aqueous phkise rcvstractecl with an qua1 volume of water saturated phenol for 10 min at, 4”. The RKA was precipitated with 2 1701of ethnnol and 0.1 vol of 20%. sodium acetate at, -20” for 2 hr, and was collcctctl by cc,ntrifugntioii at 2000
The preparation of PLK columns was l~ascd on the method described and Blamine (61 and was carried out. as follows: Kieselguhr (10 gim) was washcadextensively ill dciollizrd water ati(l w-ns suspenderl in 65 ml of water; 40 ml of thi s Fuspension was hoilell to fspcl air, and. hy Ayad

280

BHOMLEY

AND

BARRY

after cooling, 4 mg of poly-L-lysine in 0.5 ml of water n:as added and the mixture was shaken vigorously. This preparation of PLK can be stored indefinitely at room temperature. A standard column of PLK consists of four layers in a 1 cm sintcredglass tube, each layer being packed under air pressure with an air pump (type DYMKl, Charles Austen Pumps Ltd., High Road, Byfleet, Surrey, England). The bottom layer consisted of 1 ml of cellulose powder in water; the remaining layers consisted of 0.5 ml of washed kieselguhr, 7 ml of PLK, and 0.5 ml of washed kieselguhr. Each layer was packed under air pressure, with care being taken to avoid drying of the column while packing. The column was routinely washed with 25 ml of 0.4 Al NaCl. Loading

the Column

1 to 2 mg of RNA was dissolved in 9 ml of 0.4M NaCI, pipctted onto the top of the packed column, and loaded by pumping t.he solution through the column under air pressure at a flow rate of about 40 ml/hr. After loading, the column was washed with a further 35 ml of 0.4 111 NaCl. Sucrose Density

Gradient

Analysis

Linear 5-20s sucrose gradients (5 ml), containing 0.01 M Tris, 0.001 M EDTA, and 0.1 M NaCl, were centrifuged at 50!000 rpm for 2.5 hr at 4°C in a Beckman Spinco SW50 rotor. Gradient:: w:(trc fractionated by collecting drops from the bottom of the tube, and the individual fractions were assayed for RNA content. Estimation

of RNA

3H-Labeled RNA was estimated by liquid .sicintillation counting. 0.,5 ml of each column fraction was added to IO ml of Bray scintillntion fluid (8), and was counted for tritium in a Packard Tri-(‘nrb liquitl scintillation counter, series 4000. Density gra#dicnt fractions were made up to 0.5 ml wit,h deionized water and were counted in the same way. Where possible, the concentration of RNA was also determined optically by measuring the absorbance of each sample at 260 rnp in a Unicam UV spectrophotometer, SP 500. RESULTS

Assembly

and Calibration

of PLK

Column for Gradient

Elution

The apparatus used for the gradient elution of RNA from PLK columns was prepared as follows: The column was packed into a 1 cm

FRACTIONATION

OF RNA

ON

281

PLK

sintered-glass t’ube and connected to the first of two 60 ml Quickfit separating funnels, placed one above the other. The lower chamber contained 0.4 M N’aCl, and the upper chamber 4.0 M NaCl. All connections were by means of ground-glass joints. A still head with a side arm connection was situated above the upper chamber. Pressure was applied to t’he column through this side arm by means of the air pump. The air pressure was controlled by means of a screw clip fixed after a T joint connection between the pump and the side arm. Mixing in the lower chamber was effected by a steel paddle, the shaft of which passed through both reservoir chambers, and was connected to a motor suspended above the still head. During operation the paddle rotated rapidly. An air seal was provided by a drilled nylon stopper in t,he top of the still head, and this 8slso acted as a guide for the stirring paddle. From the bottom of the column, Teflon tubing led via a syringe needle to a fraction collector. As a rule, 0.7 ml fractions were collected. To check the efficiency of mixing in t,he gradient forming apparatus, and to estimate t,he molarity of NaCl leaving the column, 3H-uridine was added to the 4.0M NaCl in the upper chamb’er. Mixing was begun and the efficiency of gradient formation was determined by measuring the amount of ‘H-uridine in successive fractions of the column effluent. A close correlation was observed between the experimentally measured F;radJ!ent and the theoretical gradient, calculated using the formula: c* = (I., -

(& I - (‘,)r--v,‘r’O

(1)

where C is the concentration of 3H-uridine in the effluent from the lower chamber to the column and C2 ‘and C, are the concentrations in the upper and lower chambers, respectively. V, is the volume of the lower chamber, and ‘V the volume of the effluent at any time. This indicates that efficient mixing occurred in the lower chamber of this apparatus.

Fractionation

of Chick Embryo

RNA

on Columns

of PLK

Chick embryo cell (CEF) RNA was fractionated on a sucrose density grad:ient (Fig. 1). The gradient profile contained the typical 18 S and 28 S ribosomal RNA peaks in both the optical density and the radioactivity tracings as well as a broad 4-5 S soluble RNA peak. When a sample of this RNA was loaded onto a standard PLK column and subjected to salt gradient elution, four main species were eluted sequentially from the column, as shown in Figure 2. These peaks, designated PI to P,, were eluted at the following salt molarities: P, eluted matl.OM NaCl, P, eluted at 1.3M NaCI, P, eluted at 1.6M NaCI, P4 eluted at 1.9 M NaCI. The elution profile of CEF RNA on salt gradient elution from a PLK

282

0.3

I 0.2 iz 4

0.1

/

l‘f ,-.-.-0-e.

0 0

4

8

FIG. 1. Sucrose density gradknt at 50,000 rpnl for 2.5 hr nt 4°C 0.01 h1 Tris, and 0.001 ill EDTA. in the bottom of the centrifuge for RXA content by measuring ing for tritium (closed circles). bottom of the tube arc> plotted

12 Fraction

16

20

24

28

:m:rlq.sis of CEE’ RNA. CEF RNA was centrifuged on a 5-20 r/b sucrose grudient containing 0.1 hl NaCl. The gradient was frnrtionated by piercing a hole tube and collecting drops. Fractions were assayed ahsorbnncp at 260 mp (open rircles), and by countThe values for gradient fractions collected from the from left to right.

column shows good separation of peaks 1 and 2, but rather poorer definition of peaks 3 and 4. In an attempt to improve the definition of the fractionation procedure it was decided t’o elute RNA4 from the column with discrete steps of increasing salt molarity (stepwise elution), in place of the increasing salt gradient previously employed. The molarities of the steps chosen were those stated in “Methods.” For the stepwise elution procedure the still head was connect.ed directly to the column and the drilled nylon st.opper replaced by a ground-glass stopper. Each molarity step, starting from 0.7 M NaCl, was pipetted gently onto the t’op of a loaded column, and approximately 15 ml was run through the column under air pressure to give a flow rate of 30 ml/hr. Each step was run until the meniscus of the loading solution touched the surface of t,he column.

283

FIG. 3. The strpwise rlution of CEF RSA from a PIX column. CEF RNA was loaded to a PLK column and W:IS rlutwl by pawing dincrete molarity steps of NaCl through the column under air pwxsurc, to give :L flow rate of 30 ml/hr. Each molarit) step (15 ml) was run until the meniscus of the loading solution touched the surInce of the column. RNA content of t,he fractions was estimated by counting for tritium (open circles) and the NaCl molnrit,y st.cps c>mploped are indicakd between t,he arrows at the bottom of tlw figure. P,-I’, rrfer to the four pcnks of RNA PhIted from thj> column at, proprrssixvly higher salt molarit? SIC~~<.

284

BRUMLET

AND

ISAKRT

with the results of the gradient elut,ion. The resolution ohtaincd by stcsl)wise eIut.ion is very much grcat,er than that, obt:rinc(l OII glnclicnt olution of the column. In the cast: of stepwise rlution of tllca RK\;A, rach sl)(Jcics is eluted within the first two or three fractions of tsach molarity step.

To charact,crize the four main species of RNA eluted from the PIX column, the peak samples of each of the four RNA fractions were dialyzed against deionized water and t,he RNA precipitated in the presence of carrier yeast RISA, as described in “Methoda.” Figure 4 shows the result of sucrose density gradic~nt analysis of the four rel)rc~cipitatetl

FIG. 4. Sucrose density gradient characterization of the RNA peaks PI-P, clutcd from a standard PLK column. The four reprccipitatcd RN.4 fractions, ohtainrd b> eluting CEF RNA from a PLK column, were centrifuged at 50,000 rpm for 2.5 hl at 4°C on 5-20% sucrose gradients containing 0.1 M RaCl, 0.01 M Tris, and 0.001 31 EDTA. Gradients were fractionated by collecting drops from the bottom of the tube, and individual fractions were assayed for RXA content by counting for tritium (open circles), as described previously. A to D show sucrose density gradient annlyses of the column RNA pea,ks PI to P,, respectively. The position of the 28, 18, and 4s RNA markers are taken from sucrose density gradient analyses of unfrxtionatr>d CEF RN,4 run under identical condition>.

FR.4CTIONATIOh-

OF

RN.4

OS

PLK

285

RNA fractions. The ljositions of the 28, 18, and 4 S RN,4 are taken from gradients of thr unfractionntt>tl RNA run under ident,ical conditions. Thf, RN,% of peak 1 (Fig. 4,4), which is the first to he eluted from the column, ran on sucrose density gradient cent,rifugation as a discrete 4 S I)nn~!, and contained no RN*4 of higher sedimentation coefficient. The second peak of RNlZ elutcrl from the column (Fig. 4B) consisted of a mixture of 4 8 RNA and RKA of a slight,ly higher (C-8 S) sedimentation vnh~c*. Tht> third peak of RKA (Fig. 4C), consist,ed cnt,irely of 18 S ril~o~omal RWA. The last peak of RN,4 elut,cd from the column (Fig. 4D), althol~gh cluting as a single sharp peak from thr column, appeared on sucro::c density gradient analysis to be a mixture of 18 and 28 S rihosomal II NA. Thee re.qults suggest that, ccl1 transfer RNA. has been fractionated into ncvcral compont~nt.s on the column (P, and P,). It also appears that, un~ler the ionic contlitionr; used for elution of RNA from the column! the I~roportionnl separation of rihosomnl RKA into 18 and 28 S species is quite diff’erent from that obtained on sucrose density gradients alone! whcrc> the ionic strc>ngth of the solut,ion is much lower. On the column, a, proportion of the l&S RNA (P3) is elut#ed at a lower salt molarity than the bulk of the ribo~omnl R?U’A (P,) ; t,his RN.4 may be chemically modiCccl it1 zorncl way. DIscussIoN

The poly-r,-lyrist: coated kiesclguhr column is essentially an anioncscha:lgc column with the amino groups of t#he poly-L-lysine acting as ion-cschnnge sites. Poly-L-lpsine at. neut,ral pH has a high positive charge>, and might reasonably b’e expected to interact. with the negative rhargtx of t.hc phosphate groups of t#hc RNA molecule. By raising t’he sotliurn t~hloriclt~ nonceut,r:ttion of the column eluent,, RNA molecules arc rt~l(~as:~cl from thcl column. This reduction in t)he stability of the RNA/ poly-I,-Iysine complcs mhcn the ionic strength of the column eluent is raisctl pugpest,+ that ionic bonding of RNA t,o poly-L-lysine is an important part of their interaction. In addition, the binding of RNA to colurnn~ of l,oly-r>-lysine may involve hydrogrn bonding, and would be st.ruct,urc of the molecule. Regions of basr cltycmlt~iit. on the secondary l’airing in the molcclllc would also rc$trict t,ht> formation of additional 11ydrogcn bonds to the column. It i:; therefore to hc espccted t’hwt the elution profile of an RNA species frotn tht> colurnI1 will tlepcntl on tll(l size, secondary stVructurc, and base tLoml)osition of the RNA molecules. The t,lution of R.NA would vary with t~hangc~s ill the pH, ionic st,rength, ,antl temperature of the column elucnt, all of which would affect, thcb unique, folded structure of t’he RNA

molecules, and thus affect’ the drgrw and type of hontling bctwern the RKA and the poly-L-lysinv. Somc~ data on t’litb iraturc of the I)in(ling of nuclrotides to poly-L-lysine has hrcw proxGl(~cl 1)~ I,acq :tncl Pruitt ( 9) . They invcst,igated the intcractions l~ctween nio~ioril~oi~r~cl~~oti~l~~~nn(l solutions of poly-L-lysinc of molecular weight about 100,000. and foun~l that all the mononucleotidcs cwcl)t uritlylic acitl (lvlIP1 producetl turhidit’y in solution, while nuclrosidw, lacking thv neg:tti\-cly charged phoq~l~atc~ groups, did not. The capacity for intlucing turhitlity n-w in tlic order: G-\IP > AIMP > C:JIP > TJIP. It was collcludccl that the increaw in turbiclity is probably due to a two-stcl) l)roceah involving: ((11 ionic binding of the mononucleotides to the ljoly-I,-lysine chains, followed 1)s (b) interchain nuclcotid~~nuclvotidc interaction:: of the houncl nuclvotides. It, appears that thr production of turbidity is the wsult of slwcific iiucl(lotidc-nuclcotidc interactions. The binding of large RNA n~olcculcs to columns containing polp-Llysinc is prohahl\- dependent. on the combination of ionic bonding of inclividual nuclcoti~les, availability of hytlrogcn bonding, ancl intcrchxin nuclcotide-nucleoti(le interactions. In t)his way the specificity of clution mill he influcwcc(l by size. 1~:~ composition, ant1 sccondwrp structjurr of the RNA molcculcs. The use of PI,I< coluuuls inwituhly leatla to I compariron with thca standard us(’ of RIAK columns for thcl fractionation of RNA. The rclativcly greater chxgc on poly-L-lysine compred to t’liat of mcthylate~l serum albumin at neutral pH gi\ 73s c rise to stronger hintling of nucleic acids, as well as :I grvatcr capacity for them. The Ftronger binding can he swn from a comlw?son of tliv NaCl molaritics required to clutr various Rh’Ae from the two columns. Sucoka. and Tnmanr~ (4) 11:ive ohrequired to clutc tainetl the following \-alucs for the NaC’l molnritics Escherichirc roll: RSA from MAR columns: 4 8 RNA eluted at 0.45 J1 NaCl, 16 S RNA clutcd at, O.SJJ NaCl, and 23 S RNA eluterl at 0.9 31 NaCl. Our results using CEF RKA on PLK columns gave the following correspondingly higher values: 4 P RXA eluted at 1.0 31 NaCl, 18 S RNA rluted at 1.6 M NaCl, and 28 S RNA cluted at’ 1.9 dl NaCl. 9t neutral pH the higher tlegrce of ionization of poly-L-lysine givw rise to many more sites for the initial binding of nucleic acids than in the case of methylated wrum nll~umin. It has been found possible to loatl much larger yunntitiee of RNA to PLK colunnla than to MAK columns of comparal,le size. This higher capacity offers an obvious advantage of the PLJX column. JVe cannot c~sclurl(~ the ~xwsibility that sonic’ species of R?;X may biiicl (cf. 11AK columns (10. 11 I 1 l)utj ii) tllcwi irrerersihly with poly-r,-lysiiicx