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Fractionation by column chromatography in aqueous methanol of partial Ustilago sphaerogena nuclease digests of RNA
Fractionation by Column Methanol of Partial Nuclease
Chromatography in Aqueous Ustilago sphaerogena Digests
of RNA
LTstilago sphfreroyena RNase U, exhibits a J~rc~fercwce, rather than an absolute specificity, for the hydrolysis of purine phosl~hocliester linkages in RNA (l-3). Thud, in contrast to the compounds derived from RNA found by the action of pancreatic Rn’arc or RNase T,, the trinucleoticles in RNase U, digests (4) cliffer from each other not only by purine-pyrimidine ratios (e.g., Cl)Al)L~l~ w CIlQAp), l)ut also by Gp content (CpApGp vs CpGpGp) .I R?;nsc IT2 digests may bc resolved according to chain length by ion-exchange chromatography on DEAE-Sephades in 7 M urea (5,6). Howcvcr, further bubfractionation of the trimer mixture at pH values in thr p1< range of the NH, groups of Cl), Al), and Gp (7) failed to yield single compounds, a result obtainnble nit11 either pancreatic RNaee or RNase ‘I’, digests (8). Tllis is not surl)rising since the latter mixtures contain 8 and 9 trinuclcotides, rcsIwtively, while the RNaac U, digests may cotltain more t,han 15 trinlcrs (41. Because of the problems involved in the resolution of RKnsc U, digests of RNA at the preparative level, ion-cscllange clironintograJ)li~ systems coniplcmentary to these containing 7X urea were investigated. In this paper, the effects of substituting 5O’;lrMeOH for 7 111 urea in DEAE-Sephadex chromatography was esamincd at two pH values, using eneymic digest’s of RNA prepared with R.Xas;c IT,, pancreatic RNase, or RNase T,. At neutral pH and in 50% MeOH, RNase T, digests were resolved according t,o chain length, &hile RNwe IT2 and pancreatic RNase digests were not. The result’s indicate t.hat’ resolution of trimer mixtures by column chromntogr:~J~hy with 50% ~IcOH reso1vc.i; oligomera not only 1 While our enzyme d
strain of Ustilago sphcrerogena vwy similnr prolwrtirs and
are
than con-
OIL the basis of net charge: but also OII the i~~in~l)(sr of Gp residues 1~1 cwr~ym~~ntl. \Vhilc furth(v. ir~ic~tioli:~timr oi tlic trini(W from RNasc U, cligests in 7 M urea clicl not yicltl single c~mpou~~d:: at l)H 3.3, rcchromatography on DEAE-Sephxclc~s in 50$ McOH at the same l)H did produce the desired results. MATERIALS
AND
METHODS
All spectrophotometric measurements were made in cells with a 1 cm light path and are expressed as ahsorbancy (il). For paper chromatography with Whatman No. 3 paper, two solvents were used: solvent ,4 (n-propanol/concentratcd KH,OH/HZO 55:10:35 by volume) and solvent, H (40 gm (NH,),PO, added to 100 ml 0.1 M Tris HCl, pH 7.5). High molecular weight’ RXA from yeast was prcparcd by the proccdnre of Crestfield et nl. (9), and the fraction insoluble in 1 M NaCl was extracted twice with phenol. RNA of bacteriophage BIS2 was ohtamed as reported (10). DEAE-Pcphadcx A-25 (1 meq/gm) xas obtained from Pharmacia, and DEhE-cellulose (0.8 meq/gm) from Schleicher and Schuell, Keene, K. H. Enzy?)~. Alkaline phosphatase from Escherichk coli (Sigma, type III) was further purified to ensure removal of nucleases, (11) and the purified enzyme was used for tlcphosphorylation (12). Pancreatic R.Nase 1type III A) was obtained from Sigma. RNase T, and T, were isolated from Sanzyme (13,14), and U, RNase from I’stilngo sphneroyem (ATCC 12421)
(4).
Enzy,~zic digests. Partial RNase U, digests of RNA were prepared with 10 units of enzyme per milligram of sub&rate, corresponding to stage 2 of enzymic hydrolysis (4). IJmit digests of RNA with pancreat.ic RNase and RNaee T, were obtained as described (14, 15). Acid hydrolysis of 2’,3’-cyclic terminal phosphate residues to the corresponding 2’- and 3’-phosphate form was by 0.1 AT HCI at 23” for 3 hr. The solutions were then neutralized and desalted on DEAE-cellulose (16). Column chromatography. To prevent contaminat’ion of ion exchangers with nucleases, all enzymic digests were extracted wit’11phenol (7). Columns were packed by gravity (DEAE-Sephadex), or with a linear pressure gratlicnt to 15 psi (DEAE-cellulose). The adsorbent in t,he columns was then equilibrated with atart,ing buffer (Tahlr 1 ) until the pH and condurtioity of infloming and outflowing buffer were the same. All pH values were determined without dilution, i.e., in 50% MeOH or in 7M IIW~. The columns were kept at 23” and run at a slower flow rate than t,hat obtained with an open system. After each experiment, the adsorbent
FR.\(“flllS.\1‘IOS
01;
KS.\
l)l(IES’f~
.i3i
i: w(‘rc repacked. Conditions extrucled mtl \\‘8811e3(1, alld tdle wllunll. for column rhromatography WC given in Table 1. Characterizntion 01 VLOHO- and oliyontlcclotidc frtrctiorls. XIaterial elutetl from columns developed with 7 M IIIY’~ were dcsaltcd on DEAEcellulose (16). When buffers cont.aining 50% MeOH and ammonium formatc w(lrc usetl, McOH n-a:: rc~movc~tlc~ithcr 1);~flash C~val)oration at 30” or by repeated extraction wit,h 2 vol ether. Solutions were then freed of ammonium formate by lyophilization using a high-capacit’y vacuum pump. As an alternative to removing methanol first, 1 vol tert-butanol was added to the pooled eluate so that it could bc frozen and the material was then lyophilized directly. No loss or degradation of trimers was observed during the removal of MeOH and volatile buffer by these methods. The desalted compounds were then examined by mapping (14,15), and identified by: spect,rophotomet,ry at pH 2 and 7, treatment with pancreatic ribonuclease, RNase T, or RNase T, and resolution of products by dephosphorylation, remapping, or paper chromatography with solvents A or B. The proportion of 2’,3’-cyclic terminal phosphate and 3’-phosphate forms of mono-, di-, and tr inucleotide;; was obtained by paper chromatography at pH 7.5 with solvent B. As an example, the characterization of the clinuclcotidc (;pGp will be described in detail. This compound is part’icularly intractable and may even be lost by crystallization on beaker walls (17). To avoid possible solubility problems, the material from peak 5, Fig. 7, was desalted on DEAE-cellulose with a recovery (on t’he basis of ;I,,;,) of 78%. Since the compound was largely present in t,he 2’,3’-cyclic terminal phosphate form, it was treated with 0.1 N HCl for 3 hr at 23” to obtain the corresponding 2’-and 3’-phosphate forms. The solut,ion was adjusted to pH 8.0 with solid Tris and trentrd with an PSCW::of alkaline phosphata~c, 2500 units enzyme/mg substrat#e(18), for 3 Irr at 37”. The reaction product was freed of phosphatase by paper chromatogral)hy with solvent1 B, eluted with water, and treated with RXasc T, I 1 unit/mg substrate) in 0.1 M sodium acetat#e,pH 4.0, for 3 hr at 37”. By I)apcr chromatography with solvent B, only Gp and guanosine were found in the ratio of Gp./ guanosine of 0.91, in agreement with the postulated sequence of GpGp. was
RESULTS Yeast and MS2 RNA were partially hydrolyzed with RNase U,. Most of the resulting oligonucleotides terminated in Ap or Gp. Ap and Gp were the only mononucleotides present and the proportion of 2’,3’-cyclic terminal phosphate:3’-phosphate forms was roughly 1: 1. The RXA digests were then fractionated according to chain length (net charge) up to the heptamer level by chromatography on DEAE-Sephadex A-25 in
FRACTION
NO.
FIN:. 1. Chromatography of RNase U: digest of yeast RNA on DEAE-Sephadex A-25 in 7 ilf urea/sodium acetate, pH 5.2. Peak 1 contained Ap and Gp, and peaks Z-7 contained di- to heptannclc~otides, respectively. For column chromatography conditions and identification of compolmds. SW Table 1 and text.
7M urea with linear gradients of sodium acetate at pH 5.2 (Fig. l), With t.he same system but at pH 7.5 (5,6), t,he presence of cyclic terminal phosphate forms resulted in multiple peaks for compounds of the same chain length. Similar results were obtained with either yeast or MS2 RNA. The trinuclcotides so obtained were further resolved by chromatography on DEAF,-cellulose and DEAE-Sephades in 7111 urea at pH 3.3. About twice as many fract.ions were obtained with DEAE-cellulose (Fig. 2) as with DEAE-Sephades (not shown). On DEAE-cellulose, most of the peaks contained mixtures of trinuclcotides such as: UpUpCp,CpGpGp, and UpUpGp,UpGpGp. Further (CPUP) GP ; CPAPGP,(UPCP)AP; fractionation of such mixtures was carried out on DEAE-Sephadex by
540
FRACTION
E’lc:. 3. Fractionation of mixture of UpUpCp b\- column chromatography on DUE-Scphadcs U,IU~G~ ; peak 2. UpGpGp. For conditions T:kk 1 and test.
NO.
and I~~K~~IG~~ (see leak 12! Fig. 2) in 5O
substituting 50% MeOH for 7 121 urea. UpUpGp could thus be reparated from UpGpGp (Fig. 3). With a composite sample containing the major trinucleot’icles from RNase U, digests, (Fig. 2 1, t,hc same type of column chromatography with 50% MeOH on DEAE-Sephades at pH 3.3 also produced a series of mixed peaks (Fig. 4)) but their composition differed from those obtained on DEAE-cellulose with 7 X urea at the same pH 8
0.6
ay
0.2 0
loo
200
300
400
CJ Qifi
12 06 0
FIN:. 7. Column
chromatography
100
200
(Table
300
400
FRACTION
NO.
FRACTION
NO.
1) of pnrii:~l
On the basis of various tests, Tomlinson and Tenor (5,ci) reported that aqueous methanol would not sufIice t,o remove all secondary binding forces involved in column chromatography of small oliginucleotides on DEAE-cellulose. Later it was shown that thcrc was less secondary binding of oligonucleotides to DEAE-Sephadex t’han to DEAE-ccllulosc. Because oligonucleotidc fractions are more readily freed of incthanol and ammonium formate than of 7 M urea or inorganic salt soliitions (19)) the MeOH system was reinvestigated with DISAE-Sephadcx. The results indicate that methanol can rel)lacc 7 31 urea iii the fract,ionation of small oligomcrs according to ilet) charge provided that each compound contains only one Gp reridue as found in RNase T, cligests of RNA. By contrast, the elution patterns obtained with pancreatic and RKase U, digests show that compounds of equal chain length but different Gp content are not resolved only according to net chitrgc in 50% hIcOH. The alcohol affects hydrophobic bonds more than hydrogen bonds, while 7 M urea disrupts both forms of xecondary billding (5). This may indicate that’, in 50% &OH, Gp rontribuks more to hydrogen bonding t,han t’he other bases. The use of aqueous mcthauol ~1111s volatile buffers should complement, 7 L\{ urea solutions for the column ~hl~OlIl~~O~~~~J~h~ of such small and possibly larger oligonucleotidcs.
The major trinucleotides in partial Cstilac~o sphneroyerm RKase U, cligests of yeast and MS2 RNA were fractionated by column chromatography on DEAE-Sephadex or DEAE-cellulore in 7 JJ urea and/or in 5070 methanol on DEAE-Sepha(les at. neutral and acid $3. The 50% methanol system at. neutral pH rcsolvctl RNase T, limit dig&b of RN,4 according to chain length up to the l~cl~tanucleoti~lelcvcl. Pancreatic and Ustilaqo ribonuclease digesk were similarly frnkonated up to the trimrr 1~~1, not only by chain length but also according to Gl) content I)er compound. HEFERKNCEG 1. AKIMA. 2. UCHIDA, 3. ADAMS. 1009
T.. UCFIID.A. T.. AND Ecaw, I;.. I3iocherr~. J. 106, 601 (1968). T.. ARIMA, T., AND EGAMI, F.. J. B&hem. (Tokyo) 67, 91 (1970). J. M.! JEPPRSEN, P. G. N., SANGER, F.. AND B.IRREL. B. G., Xatlrre 223, (1969). 4. RUSHIZKY, G. W.. MOZEJICO. J. H., ROGEWON, II. T,., JR., AND SOHER, H. A.: Biochrmb/r?/, 9, 4966 I 1970)
R. V., AND TEN&R: G. M., J. Amer. Chem. SOP. 84, 2644 (1962). R. V.. ANI) TENEK. G. M., Biochemislry 2, 697-703 (1963). RUSHIZKY, G. W., BAHTOS, E. M., AND SOBER. H. A., Biochemistry 3, 626 (1064). 5. RUSHIZKY. G. W., IND SOBER, I-1. A., Biochrm. Biophys. Res. Comnwrt. 14. 276 (1964). 0. CRESTFIELD. X. M., SMITII, K. C.. .43-D IZLLEN. F. W., J. Rid. Chew. 216, 185, 5.
TOMLINSON,
6. 7.
TOMLINSON.
(195.5). 10. 11. 12. 13.
d. H., JR..
STRAWS,
WMRS. ?JEL-,
LIVE.
R..
H.
~
C., ,?.,
T.
.xxD AS,,
.wD
R.,
HEPPEI.. ~.\I<.\MI;R.4,
R. L., J. Nol. Biol. 7, 43 (1963). C. C., J. Bid. Chem. 243, 4530 (196s). L. 9., J. Bid. Chem. 240, 3685 (1965). I\.. “hIicd,inl Nihnu~lrasrs,” p. 1s. Spl,ill~l’1.-~.,,1.1:1~$.
SINSHEIMER. AND
RICHARDSON,
New York. 1969. 14. 15. 16.
17. 18. 19.
Sotlm. H. A.. J. Hid. Chum. 237, 534 (1962) ~NIGII’I’. c. a.. T-irdogy 11, 236 (1960). RUSIIIZKY. G. Jt-., .ISI) SOw,ft. H. 3.. Biochim. Biophys. Actn 55, 2li (l!l62), S~XSIIEIMER. I<. I,.. .I. Biol. (‘hum. 215, 579 (1955). 410x. B. N.. ASI) I~-BIx. D. T.. J. Viol. (‘hcnl. 235. 769 (1960). Corrx. W. E., it1 “Cll~~on~nic~rrr~~~l~~" (TC. Hvfl mm. cd.). p. 554. Iicidlr~lrl. SC\\ \IeOrli. 1961. 11rrsa1zr~r.
G.
m’..
ASD
~~SIII%KY,
c;.
I!-,,
ASD
Chromatography in Aqueous Ustilago sphaerogena Digests
of RNA
LTstilago sphfreroyena RNase U, exhibits a J~rc~fercwce, rather than an absolute specificity, for the hydrolysis of purine phosl~hocliester linkages in RNA (l-3). Thud, in contrast to the compounds derived from RNA found by the action of pancreatic Rn’arc or RNase T,, the trinucleoticles in RNase U, digests (4) cliffer from each other not only by purine-pyrimidine ratios (e.g., Cl)Al)L~l~ w CIlQAp), l)ut also by Gp content (CpApGp vs CpGpGp) .I R?;nsc IT2 digests may bc resolved according to chain length by ion-exchange chromatography on DEAE-Sephades in 7 M urea (5,6). Howcvcr, further bubfractionation of the trimer mixture at pH values in thr p1< range of the NH, groups of Cl), Al), and Gp (7) failed to yield single compounds, a result obtainnble nit11 either pancreatic RNaee or RNase ‘I’, digests (8). Tllis is not surl)rising since the latter mixtures contain 8 and 9 trinuclcotides, rcsIwtively, while the RNaac U, digests may cotltain more t,han 15 trinlcrs (41. Because of the problems involved in the resolution of RKnsc U, digests of RNA at the preparative level, ion-cscllange clironintograJ)li~ systems coniplcmentary to these containing 7X urea were investigated. In this paper, the effects of substituting 5O’;lrMeOH for 7 111 urea in DEAE-Sephadex chromatography was esamincd at two pH values, using eneymic digest’s of RNA prepared with R.Xas;c IT,, pancreatic RNase, or RNase T,. At neutral pH and in 50% MeOH, RNase T, digests were resolved according t,o chain length, &hile RNwe IT2 and pancreatic RNase digests were not. The result’s indicate t.hat’ resolution of trimer mixtures by column chromntogr:~J~hy with 50% ~IcOH reso1vc.i; oligomera not only 1 While our enzyme d
strain of Ustilago sphcrerogena vwy similnr prolwrtirs and
are
than con-
OIL the basis of net charge: but also OII the i~~in~l)(sr of Gp residues 1~1 cwr~ym~~ntl. \Vhilc furth(v. ir~ic~tioli:~timr oi tlic trini(W from RNasc U, cligests in 7 M urea clicl not yicltl single c~mpou~~d:: at l)H 3.3, rcchromatography on DEAE-Sephxclc~s in 50$ McOH at the same l)H did produce the desired results. MATERIALS
AND
METHODS
All spectrophotometric measurements were made in cells with a 1 cm light path and are expressed as ahsorbancy (il). For paper chromatography with Whatman No. 3 paper, two solvents were used: solvent ,4 (n-propanol/concentratcd KH,OH/HZO 55:10:35 by volume) and solvent, H (40 gm (NH,),PO, added to 100 ml 0.1 M Tris HCl, pH 7.5). High molecular weight’ RXA from yeast was prcparcd by the proccdnre of Crestfield et nl. (9), and the fraction insoluble in 1 M NaCl was extracted twice with phenol. RNA of bacteriophage BIS2 was ohtamed as reported (10). DEAE-Pcphadcx A-25 (1 meq/gm) xas obtained from Pharmacia, and DEhE-cellulose (0.8 meq/gm) from Schleicher and Schuell, Keene, K. H. Enzy?)~. Alkaline phosphatase from Escherichk coli (Sigma, type III) was further purified to ensure removal of nucleases, (11) and the purified enzyme was used for tlcphosphorylation (12). Pancreatic R.Nase 1type III A) was obtained from Sigma. RNase T, and T, were isolated from Sanzyme (13,14), and U, RNase from I’stilngo sphneroyem (ATCC 12421)
(4).
Enzy,~zic digests. Partial RNase U, digests of RNA were prepared with 10 units of enzyme per milligram of sub&rate, corresponding to stage 2 of enzymic hydrolysis (4). IJmit digests of RNA with pancreat.ic RNase and RNaee T, were obtained as described (14, 15). Acid hydrolysis of 2’,3’-cyclic terminal phosphate residues to the corresponding 2’- and 3’-phosphate form was by 0.1 AT HCI at 23” for 3 hr. The solutions were then neutralized and desalted on DEAE-cellulose (16). Column chromatography. To prevent contaminat’ion of ion exchangers with nucleases, all enzymic digests were extracted wit’11phenol (7). Columns were packed by gravity (DEAE-Sephadex), or with a linear pressure gratlicnt to 15 psi (DEAE-cellulose). The adsorbent in t,he columns was then equilibrated with atart,ing buffer (Tahlr 1 ) until the pH and condurtioity of infloming and outflowing buffer were the same. All pH values were determined without dilution, i.e., in 50% MeOH or in 7M IIW~. The columns were kept at 23” and run at a slower flow rate than t,hat obtained with an open system. After each experiment, the adsorbent
FR.\(“flllS.\1‘IOS
01;
KS.\
l)l(IES’f~
.i3i
i: w(‘rc repacked. Conditions extrucled mtl \\‘8811e3(1, alld tdle wllunll. for column rhromatography WC given in Table 1. Characterizntion 01 VLOHO- and oliyontlcclotidc frtrctiorls. XIaterial elutetl from columns developed with 7 M IIIY’~ were dcsaltcd on DEAEcellulose (16). When buffers cont.aining 50% MeOH and ammonium formatc w(lrc usetl, McOH n-a:: rc~movc~tlc~ithcr 1);~flash C~val)oration at 30” or by repeated extraction wit,h 2 vol ether. Solutions were then freed of ammonium formate by lyophilization using a high-capacit’y vacuum pump. As an alternative to removing methanol first, 1 vol tert-butanol was added to the pooled eluate so that it could bc frozen and the material was then lyophilized directly. No loss or degradation of trimers was observed during the removal of MeOH and volatile buffer by these methods. The desalted compounds were then examined by mapping (14,15), and identified by: spect,rophotomet,ry at pH 2 and 7, treatment with pancreatic ribonuclease, RNase T, or RNase T, and resolution of products by dephosphorylation, remapping, or paper chromatography with solvents A or B. The proportion of 2’,3’-cyclic terminal phosphate and 3’-phosphate forms of mono-, di-, and tr inucleotide;; was obtained by paper chromatography at pH 7.5 with solvent B. As an example, the characterization of the clinuclcotidc (;pGp will be described in detail. This compound is part’icularly intractable and may even be lost by crystallization on beaker walls (17). To avoid possible solubility problems, the material from peak 5, Fig. 7, was desalted on DEAE-cellulose with a recovery (on t’he basis of ;I,,;,) of 78%. Since the compound was largely present in t,he 2’,3’-cyclic terminal phosphate form, it was treated with 0.1 N HCl for 3 hr at 23” to obtain the corresponding 2’-and 3’-phosphate forms. The solut,ion was adjusted to pH 8.0 with solid Tris and trentrd with an PSCW::of alkaline phosphata~c, 2500 units enzyme/mg substrat#e(18), for 3 Irr at 37”. The reaction product was freed of phosphatase by paper chromatogral)hy with solvent1 B, eluted with water, and treated with RXasc T, I 1 unit/mg substrate) in 0.1 M sodium acetat#e,pH 4.0, for 3 hr at 37”. By I)apcr chromatography with solvent B, only Gp and guanosine were found in the ratio of Gp./ guanosine of 0.91, in agreement with the postulated sequence of GpGp. was
RESULTS Yeast and MS2 RNA were partially hydrolyzed with RNase U,. Most of the resulting oligonucleotides terminated in Ap or Gp. Ap and Gp were the only mononucleotides present and the proportion of 2’,3’-cyclic terminal phosphate:3’-phosphate forms was roughly 1: 1. The RXA digests were then fractionated according to chain length (net charge) up to the heptamer level by chromatography on DEAE-Sephadex A-25 in
FRACTION
NO.
FIN:. 1. Chromatography of RNase U: digest of yeast RNA on DEAE-Sephadex A-25 in 7 ilf urea/sodium acetate, pH 5.2. Peak 1 contained Ap and Gp, and peaks Z-7 contained di- to heptannclc~otides, respectively. For column chromatography conditions and identification of compolmds. SW Table 1 and text.
7M urea with linear gradients of sodium acetate at pH 5.2 (Fig. l), With t.he same system but at pH 7.5 (5,6), t,he presence of cyclic terminal phosphate forms resulted in multiple peaks for compounds of the same chain length. Similar results were obtained with either yeast or MS2 RNA. The trinuclcotides so obtained were further resolved by chromatography on DEAF,-cellulose and DEAE-Sephades in 7111 urea at pH 3.3. About twice as many fract.ions were obtained with DEAE-cellulose (Fig. 2) as with DEAE-Sephades (not shown). On DEAE-cellulose, most of the peaks contained mixtures of trinuclcotides such as: UpUpCp,CpGpGp, and UpUpGp,UpGpGp. Further (CPUP) GP ; CPAPGP,(UPCP)AP; fractionation of such mixtures was carried out on DEAE-Sephadex by
540
FRACTION
E’lc:. 3. Fractionation of mixture of UpUpCp b\- column chromatography on DUE-Scphadcs U,IU~G~ ; peak 2. UpGpGp. For conditions T:kk 1 and test.
NO.
and I~~K~~IG~~ (see leak 12! Fig. 2) in 5O
substituting 50% MeOH for 7 121 urea. UpUpGp could thus be reparated from UpGpGp (Fig. 3). With a composite sample containing the major trinucleot’icles from RNase U, digests, (Fig. 2 1, t,hc same type of column chromatography with 50% MeOH on DEAE-Sephades at pH 3.3 also produced a series of mixed peaks (Fig. 4)) but their composition differed from those obtained on DEAE-cellulose with 7 X urea at the same pH 8
0.6
ay
0.2 0
loo
200
300
400
CJ Qifi
12 06 0
FIN:. 7. Column
chromatography
100
200
(Table
300
400
FRACTION
NO.
FRACTION
NO.
1) of pnrii:~l
On the basis of various tests, Tomlinson and Tenor (5,ci) reported that aqueous methanol would not sufIice t,o remove all secondary binding forces involved in column chromatography of small oliginucleotides on DEAE-cellulose. Later it was shown that thcrc was less secondary binding of oligonucleotides to DEAE-Sephadex t’han to DEAE-ccllulosc. Because oligonucleotidc fractions are more readily freed of incthanol and ammonium formate than of 7 M urea or inorganic salt soliitions (19)) the MeOH system was reinvestigated with DISAE-Sephadcx. The results indicate that methanol can rel)lacc 7 31 urea iii the fract,ionation of small oligomcrs according to ilet) charge provided that each compound contains only one Gp reridue as found in RNase T, cligests of RNA. By contrast, the elution patterns obtained with pancreatic and RKase U, digests show that compounds of equal chain length but different Gp content are not resolved only according to net chitrgc in 50% hIcOH. The alcohol affects hydrophobic bonds more than hydrogen bonds, while 7 M urea disrupts both forms of xecondary billding (5). This may indicate that’, in 50% &OH, Gp rontribuks more to hydrogen bonding t,han t’he other bases. The use of aqueous mcthauol ~1111s volatile buffers should complement, 7 L\{ urea solutions for the column ~hl~OlIl~~O~~~~J~h~ of such small and possibly larger oligonucleotidcs.
The major trinucleotides in partial Cstilac~o sphneroyerm RKase U, cligests of yeast and MS2 RNA were fractionated by column chromatography on DEAE-Sephadex or DEAE-cellulore in 7 JJ urea and/or in 5070 methanol on DEAE-Sepha(les at. neutral and acid $3. The 50% methanol system at. neutral pH rcsolvctl RNase T, limit dig&b of RN,4 according to chain length up to the l~cl~tanucleoti~lelcvcl. Pancreatic and Ustilaqo ribonuclease digesk were similarly frnkonated up to the trimrr 1~~1, not only by chain length but also according to Gl) content I)er compound. HEFERKNCEG 1. AKIMA. 2. UCHIDA, 3. ADAMS. 1009
T.. UCFIID.A. T.. AND Ecaw, I;.. I3iocherr~. J. 106, 601 (1968). T.. ARIMA, T., AND EGAMI, F.. J. B&hem. (Tokyo) 67, 91 (1970). J. M.! JEPPRSEN, P. G. N., SANGER, F.. AND B.IRREL. B. G., Xatlrre 223, (1969). 4. RUSHIZKY, G. W.. MOZEJICO. J. H., ROGEWON, II. T,., JR., AND SOHER, H. A.: Biochrmb/r?/, 9, 4966 I 1970)
R. V., AND TEN&R: G. M., J. Amer. Chem. SOP. 84, 2644 (1962). R. V.. ANI) TENEK. G. M., Biochemislry 2, 697-703 (1963). RUSHIZKY, G. W., BAHTOS, E. M., AND SOBER. H. A., Biochemistry 3, 626 (1064). 5. RUSHIZKY. G. W., IND SOBER, I-1. A., Biochrm. Biophys. Res. Comnwrt. 14. 276 (1964). 0. CRESTFIELD. X. M., SMITII, K. C.. .43-D IZLLEN. F. W., J. Rid. Chew. 216, 185, 5.
TOMLINSON,
6. 7.
TOMLINSON.
(195.5). 10. 11. 12. 13.
d. H., JR..
STRAWS,
WMRS. ?JEL-,
LIVE.
R..
H.
~
C., ,?.,
T.
.xxD AS,,
.wD
R.,
HEPPEI.. ~.\I<.\MI;R.4,
R. L., J. Nol. Biol. 7, 43 (1963). C. C., J. Bid. Chem. 243, 4530 (196s). L. 9., J. Bid. Chem. 240, 3685 (1965). I\.. “hIicd,inl Nihnu~lrasrs,” p. 1s. Spl,ill~l’1.-~.,,1.1:1~$.
SINSHEIMER. AND
RICHARDSON,
New York. 1969. 14. 15. 16.
17. 18. 19.
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