Proc. Natl. Acad. Sci. USA Vol. 75, No. 9, pp. 4140-4144, September 1978 Biochemistry

Simplified method for purification of ribonuclease H from calf thymus (isoenzymes/isoelectric focusing/base preferences in hydrolysis/hybrid nuclease)

JANNIS G. STAVRIANOPOULOS AND ERWIN CHARGAFF Cell Chemistry Laboratory, The Roosevelt Hospital, New York, New York 10019

Contributed by Erwin Chargaff, June 2, 1978

ABSTRACT An improved purification procedure for the isolation of ribonuclease H (hybrid nuclease; RNA*DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) from the thymus of 4- to 6-months-old calves yields two highly active forms of the enzyme, designated as ribonuclease Hi and H2. Their isoelectric points are 5.0 ± 0.05 and 5.25 + 0.05, respectively; their molecular weight, estimated from gel filtration, is in both cases 64,000 + 2000. On sodium dodecyl sulfate gel electrophoresis, two principal bands were identified, with molecular weights of 32,000 and 21,000. The nature of the nucleotides at the 3'-OH terminals, produced initially by the enzymic hydrolysis of hybridized RNA, was examined and shown to be a function of the divalent metal ion employed as activator.

scribed in the same paper is in the present study designated as assay A. A second procedure, referred to here as assay B,* also has been described before (4). For the assay for ribonuclease activity of the pancreatic type, the assay mixture contained in a total of 120 Ml: 0.05 M Tris-HCl (pH 8.0), 0.02 M MgCl2, 0.1 M KC1, 20 nmol of RNA, and 15,000 units of the ribonuclease H preparation. In the controls, the enzyme was omitted. Incubation was at 350 for 90 min. The other steps were as in assay A.

Initial purification Freshly collected thymus from 4- to 6-months-old calves was transported in ice, freed of fat, and stored at -200 for no more than 2 months. The stated pH values and (NH4)2S04 saturation levels apply to a temperature of 250. Unless noted, fractionation took place at 40, centrifugation at 10,000 X g. The sequence of steps is summarized, for one run, in Table 1. Buffers. Extraction buffer: 0.05 M Tris-HCl (pH 7.8)/0.1 M LiCl/20 mM MnCI2/0.06% (vol/vol) a-monothioglycerol. Mn2+ was added to the buffer directly before tissue homogenization. Storage buffer: 0.05 M Tris-HCl (pH 7.8)/0.4 M NaCl/30 mM MgCl2, 0.5 mM dithiothreitol/50% (vol/vol) glycerol. Fractionation buffers: A, 0.05 M Tris-HCl (pH 7.8)/0.05% (vol/vol) thioglycerol. B, same as A, with 0.05 M KCL. C, same as A, with 0.08 M KCL. D, same as A, with 0.15 M KCL. E, same as A, with 0.2 M KCL. Buffers F to I were made by dilution of a 2.0 M sodium acetate solution (pH -5.4) that was prepared by adjusting 234 g of sodium acetate tetrahydrate and 17.5 ml of glacial acetic acid with water to a volume of 1 liter. F, 0.1 M sodium acetate/0.05% (vol/vol) thioglycerol. G, 0.2 M sodium acetate/0.05% (vol/vol) thioglycerol. H, same as G, with 0.05 M KCL. I, same as G, with 0.13 M KCL. J, 0.15 M glycine, 5 mM Tris base, 1.0 mM dithiothreitol; the pH was 7.7. K, same as J, with 10 mM MgCl2. L, same as J, with 10 mM Na4EDTA. Step A: Crude Extract. The still half-frozen tissue (1.6 kg) was cut to 2- to 3-cm-thick cubes and permitted to thaw at a temperature of below 5°. The tissue was minced in a blender (30 sec at low speed) with 3 vol of extraction buffer, and 15 min later 1-2 ml of octanol was added. The supernatant resulting from the centrifugation of the suspension (60 min) was filtered through several layers of glass wool. Step B: Precipitation with Polyethylene Glycol. Polyethylene glycol (120 g/liter of crude extract) was added with vigorous stirring; the mixture then was stirred slowly for 30 min and centrifuged (10 min for intermediate runs and 30 min for final run), care being taken to collect the pellets at the same cup

In a previous communication (1) we described the purification of ribonuclease H (hybrid nuclease; RNA-DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) from calf thymus. Because this is an unstable enzyme, existing in several forms (2) that possibly represent enzymically active stages in the degradation of the initial enzyme, our procedure, while giving reproducible results with the same batch of tissue, gave varying results with different samples of commercially available thymus as regards the recovery of initial activity and the specific activity of the end product. In general, the higher the activity of the crude extract, the better was the reproducibility of the procedure. Another drawback of the previous method was the fact that, for reasons of stability and final yield, only 65% of the total extractable enzyme activity could be subjected to purification. The study presented here simplifies the isolation of the enzyme and has proved quite reproducible in our hands when applied to freshly obtained thymus from 4- to 6-months-old animals. We also discuss the evidence for the existence of two forms of the enzyme, separable from each other in the crude extract.

Materials and methods In addition to the materials and their sources listed before (1), Cellex CM (CM-cellulose) was supplied by Bio-Rad, ofigo(dT)-cellulose T2 by Collaborative Research. The 3H-labeled substrates haveibeen described (1); the specific activities of their ribo moieties (cpm/nmol) were: poly(A) 2400, poly(U) 2000. The phage fi DNA- [3H]RNA hybrids with a DNA/RNA ratio of 1 (3> were in each preparation labeled with one of the four nucleotides (10,000 cpm/nmol). 3H-Labeled RNA (10,000 cpn/nmol) was prepared with the aid of Escherichia coli RNA polymerase and of calf thymus DNA as template. From 1 mg of DNA, 1.1 mg of purified RNA was obtained. The ribonuclease H unit has been defined (1); the assay deThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate

*

this fact.

4140

It should be mentioned that in this assay the step requiring complete solution of the uranyl precipitate in K2CO3 yields a homogeneous suspension if high amounts of protein are present. This does not interfere with the subsequent oxidation with m-periodate.

Biochemistry: Stavrianopoulos and Chargaff

Proc. Natl. Acad. Sci. USA 75 (1978)

4141

Table 1. Isolation of two enzyme forms: Ribonuclease H1 and H2*

Vol, Step

ml

A B C-H1 C-H2 Hi/I Hi/Il Hi/III H1/IV Hi/V H2/I H2/II H2/III

5050 1500 2000 300 300 400 10 2 3 50 7 4 5

Total protein, mg 55,300 30,484 25,000 2,502 6,960 2,000 73.8 6.9 3.0 1,000 108 24.8 4.2

Total activity, units X 10-4

Specific activity, units/mg

Yield,

4020 3513 1602 1416 1603 1363 1022 705 458 1206 846 780 530

727 1,152 641 5,659 2,303 6,815 138,482 1,021,739 1,526,667 12,060 78,333 314,516 1,261,905

100 87.4 39.8 35.2 39.9 33.9 25.4 17.5 11.4 30.0 21.0 19.4 13.2

%

H2/IV * In this experiment 1.6 kg of fresh calf thymus was processed. Assay A served for the determination of activity. One unit is defined as the quantity of enzyme converting 1 nmol of poly(dT)-poly(A) hybrid to an acid-soluble form in 15 min at 35°. When freshly obtained calf thymus, stored for no more than 2 months at -200, was processed, the results shown here for ribonuclease H2 were always reproducible. As regards ribonuclease H1, however, in two out of eight experiments the final specific activity in step Hi/V amounted only to 400,000 to 500,000 units/mg. The activity loss invariably occurred on the Cellex CM column in step Hi/II.

position. The drained sediments were transferred in portions to a 10-ml plastic syringe and extruded as a flat thread which was suspended homogeneously with vigorous stirring, in 1.5 liters of buffer B. Additional slow stirring (30 min) was followed by centrifugation (60 min). The supernatant, adjusted to 0.01 M with solid Na4EDTA, was stored overnight at 40. Steps A and B must be performed without interruption, which requires about 13 hr. Step C: Passage through CM-Sephadex; Evidence of Two Enzyme Forms. A gelatinous precipitate formed on storage was

removed (60-min centrifugation) and the solution was passed (400 ml/hr) through a column (8 X 10 cm) of CM-Sephadex equilibrated with buffer B. The column then was washed, at the same flow rate, with 500 ml of buffer B. The combined eluates, containing about half of the recoverable enzyme activity, were designated as ribonuclease Hi. The other half was eluted with buffer E (same flow rate, 50-ml fractions) and designated as ribonuclease H2. Purification of ribonuclease HI Step HI/I: Fractionation with Ammonium Sulfate. Solid (NH4)2SO4 (209 g/liter) was added to the solution of H1, and the mixture was stirred (30 min) and centrifuged (30 min). The addition of more (NH4)2SO4 (129 g/liter) precipitated the enzyme, which was, after stirring for 1 hr, collected by centrifugation (60 min). The drained pellets were dissolved in 300 ml of buffer A. Step HI/II: Fractionation with Polyethylene Glycol. The enzyme solution was diluted with buffer A to a conductivity equal to that of 0.1 M (NH4)2SO4 in buffer A. If protein absorbance at 280 nm was greater than 10, the solution was brought to this concentration with 0.1 M (NH4)2SO4 in buffer A. Following the addition of polyethylene glycol (120 g/liter), the mixture was processed as in step B. The supernatant was adjusted to 0.04 M by adding 3.0 M MgCl2, and the enzyme was precipitated by mixing 8 parts of the enzyme solution with 5 parts of 25% (vol/vol) polyethylene glycol solution in buffer A supplemented with 0.1 M (NH4)2SO4. The mixture was im-

mediately transferred to polycarbonate centrifuge bottles, kept for 30 min, and then centrifuged (60 min). The drained pellets were removed with a spatula and their solution in 200 ml of buffer G was stored overnight. Step HI/III: Fractionation with Cellex CM and CMSephadex. The enzyme solution, diluted with 200 ml of 0.05% aqueous thioglycerol, was freed of an inactive protein precipitate (30-min centrifugation) andrthe supernatant was passed though a Cellex CM column (4 X 10 cm), equilibrated with buffer F. at 150 ml/hr. The column was washed with the same buffer until the absorbance at 280 nm dropped to 0.10-0.15. The eluate, adjusted to 0.2 M by the addition of the 2 M sodium acetate solution, was applied to a CM-Sephadex column (2.5 X 15 cm), equilibrated with buffer G, which then was washed with buffer H until absorbance was down to 0.2. Subsequently, the enzyme was eluted with buffer I (40 ml/hr), 3.6-mi fractions being collected. Fractions showing no less than a 3-fold activity enrichment over the preceding step were combined, and the enzyme was precipitated with (NH4)2SO4 (390 g/liter). After 2 hr, the precipitate was collected by centrifugation (10 min), resuspended in a small amount of supernatant, and recentrifuged (35,000 X g, 60 min). The sediment was dissolved in 10 ml of buffer A. Step H1/IV: DEAE-Cellulose Fractionation. The solution, diluted with buffer A to a conductivity equal to that of 0.02 M (NH4)2SO4 in the buffer, was applied to a DEAE-cellulose column (1.2 X 10 cm) equilibrated with buffer A, which was washed with buffer B (35 ml/hr) until the absorbance (280 nm) dropped below 0.2. The enzyme was then eluted with buffer C (4 ml/hr, 0.8-ml fractions). From the combined fractions, showing a 3-fold or better enrichment, the enzyme was precipitated [390 g of (NH4)2SO4/liter]. After 2 hr the mixture was centrifuged (16,000 X g, 60 min) and the completely drained pellet was suspended (Teflon and glass homogenizer) in 5.0 ml of buffer A supplemented with (NH4AS04 (295 g/liter). Stirring for 1 hr and centrifugation (16,000 X g, 30 min) yielded a sediment that was discarded. Precipitation by additional (NH4)2SO4 (117 g/liter), stirring for 30 min, storage for 2 hr, and centrifugation (16,000 X g, 60 min) gave the enzyme pellet, which was dissolved in 2 ml of buffer K.

4142

Proc. Natl. Acad. Sci. USA 75 (1978)

Biochemistry: Stavrianopoulos and Chargaff

I0 0 0

x

-8 co 0

6 o0 0

0

HI 00 00

0 0 00 0

-7 "_ 6 E C

5'

5 00

CL

0 0

00

0 0 0 0 0

44 0

0

0

4

3

0

w,

2N

z w

I

3

55 50 45 40 35 FRACTION FIG. 1. Isoelectric focusing of a mixture of ribonuclease H1 and H2. A mixture of 1 mg each of Hi/V and H2/IV (compare Table 1) was dialyzed against four changes of 200 ml of buffer J (2 hr, 4°). Focusing was performed in the LKB apparatus 8101 at 70 for 48 hr at 500 V, with a pH gradient of 4-6. Fractions of 1.5 ml were collected and assayed with assay A.

20

25

30

Step Hh/V: Oligo(dl)-Cellulose Fractionation. The enzyme solution, diluted with buffer K to a conductivity equivalent to that of 0.04 M KCl in the buffer, was applied to the adsorbent column (0.8 X 15 cm) at a flow rate of 12 ml/hr (2-ml fractions).t Washing with buffer K was continued till the absorbance (260 nm) of the eluates fell below 0.03.$ The enzyme was then eluted with buffer L (0.5-ml fractions). The fractions showing a 1.5-fold or better enrichment were combined and applied to a DEAE-cellulose column (0.4-ml bed volume) equilibrated with buffer A. The column was washed with 0.05 M Tris.HCl (pH 7.8)/0.001 M dithiothreitol, and the enzyme was eluted with the same buffer supplemented with 0.8 M NaCl and 0.06 M Mg9l2. The first 0.2 ml was discarded, the next 0.5 ml contained the enzyme. To this eluate an equal volume of cold glycerol was added and the mixture was stored at -20° under argon. Purification of ribonuclease H2 Step H2/I: Fractionation with Ammonium Sulfate. Fractionation was performed as in step Hi/I. The precipitate was dissolved in 50 ml of buffer A. Step H2/II: DEAE-Sephadex Fractionation. The enzyme solution was diluted with buffer A to a conductivity equaling that of 0.07 M KCl in this buffer and then applied to a DEAESephadex column (2.5 X 13 cm) equilibrated with buffer B. After the column had been washed with this buffer (80 ml/hr) until the absorbance (280 nm) of the eluates was below 0.4, enzyme was eluted with buffer D (30 ml/hr, 3.5-ml fractions). Fractions exhibiting at least a 4-fold increase in activity over A suspeision of oligo(dT)-cellulose in 0.1 M KOH was washed repeatedly at room temperature by stirring, settling, and decantation. The 4urry was loaded to a column and washed with 0.1 M KOH until (260 nm) of the flowthrough fell below 0.05. The the absorbance was then washed with water to neutrality and equilibrated column / at 40 with buffer K. t Owing to the low column capacity, the late fractions of the flowthrough and the wash contained activity. The fractions were combined and readsorbed after regeneration of the column with 0.1 M KOH. t

the previous step were combined. The addition of (NH4)2SO4 (243 g/liter) with stirring produced an inactive precipitate that was centrifuged off (30 min) and discarded. Additional (NH4)2SO4 (132 g/liter) precipitated the enzyme which, after storage for 2 hr, was collected in one cup (intermediate centrifugation for 10 min, final centrifugation for 60 min, at 16,000 X g). The drained sediment was dissolved in 7 ml of buffer A. Step H2/III: Fractionation with Polyethylene Glycol. The enzyme solution was diluted with buffer A as in step H1/II, polyethylene glycol (80 g/liter) was added as in step B, and 30 min later the mixture was centrifuged (20 min). The supernatant was adjusted to 0.04 M Mg9l2 and the enzyme was precipitated with solid polyethylene glycol (90 g/liter), care being taken to dissolve the reagent by thorough agitation with a plastic rod. After 60 min the sediment was collected (60 min) and dissolved in 4 ml of buffer K. Step H2/IV: Oligo(dT)Cellulose Fractionation. The enzyme solution was in four portions fractionated as described for step H1/V. All fractions showing at least a 3-fold enrichment over the preceding step were combined, concentrated, and stored, as recorded for that step. Remarks on procedure and properties Fractionation. Polyethylene glycol, employed earlier in the fractionation of plasma proteins (5), proved its value in our preceding study (1). It has now been found that, in the presence of divalent metals, lower concentrations of the reagent suffice to precipitate ribonuclease H, thus enabling us to precipitate the enzyme quantitatively at alkaline pH. Mn2+ acted at lower concentrations than did Mg2+. The manganese salts of the oligonucleotides precipitate with the enzyme; for separation, advantage was taken of the fact that the former are insoluble in buffer B (compare step B). Because in this step the 280/260 absorbance ratio of the proteins was 1.2-1.3, no additional step for the removal of nucleic acid was necessary. Steps H1/V and H2/IV represent an application of the principle of affinity adsorption. Poly(U)-Sepharose and oligo(dT)-cellulose were compared as adsorbents; despite the

Biochemistry: Stavrianopoulos and Chargaff

Proc. Natl. Acad. Sci. USA 75 (1978)

higher capacity of the former the latter was chosen becauseit can be easily regenerated with 0.1 M KOH. Properties. The characteristics of the ribonuclease H of calf thymus have been investigated repeatedly (2, 3, 6-8). Here, we limit ourselves to the consideration of the two enzyme forms, ribonuclease H1 and H2. Isoelectric Focusing. The activity pattern of a mixture of the highly purified fractions H1 and H2 is shown in Fig. 1. The activity maximum at pH 5.0 corresponds to ribonuclease H1, that at pH 5.25 to H2. During this operation, both preparations lost more than 80% of their activity, being unstable below pH 5.4. This excluded the use of the procedure as a purification step. Effects of Divalent Cations and of Salts. The strict requirement of ribonuclease H for divalent metal ions and the differences in the susceptibility of various homopolymer hybrids as substrates have been discussed before (8). The two enzyme forms described here showed little difference with regard to the optimum metal concentration and to the activating effect of ammonium sulfate (1). Base Preference. The availablility of 1:1 fI DNA-RNA hybrid preparations (3) made it possible to go beyond the use of homopolymer hybrids as substrates (8) and to ascertain whether the nuclease, in its primary attack on a heteropolymer hybrid, showed a preference for one or the other nucleotide. It also afforded an opportunity to compare the two enzyme forms, ribonuclease H1 and H2. As can be seen in Table 2 and as was to be expected from our findings with homopolymers (8), the type of activating metal ion had a decisive influence on the nature of the terminal nucleotides released initially. Because the enzyme is an endonuclease, its initial attack on a hybridized polyribonucleotide of the type ... pNlpN2pN3pN4p ... may result, for example, in a break between N2 and N3, yielding ... pN'pN2 and pN3pN4p .... What our assay determines is the relative frequency of N2, i.e., the terminal nucleotide containing a 2',3'-diol structure, produced in the initial enzymic hydrolysis. With Mg2+ as activator, the order of preference is A > U = C > G; the differences between the two enzyme forms do not appear to be significant, with the possible exception of adenine.

4143

The- ffects of Co2+ activation are interesting: the trend of attack is G > A > U > C, with guanylic acid being by far the preponderant terminal nucleotide, especially in the case of ribonuclease H2. The previously observed (8) inactivity, and even inhibitory action, of Co2+ in the enzymic splitting of poly(dC)-poly(G) can now be explained by postulating a particularly tight binding of the Co-activated enzyme to guanine, which in the case of the homopolymer becomes unproductive, whereas in a heteropolymer a high frequency of guanylic acid breaks is favored. With Mn2+ as activator, the order of hydrolysis is A > U > C > G, although with ribonuclease H2 terminal guanine is as frequent as cytosine. Inhibition by Thiol Reagents. As reported before (1), ribonuclease H is inhibited by p-chloromercuribenzoate. Another inhibitor, but at a 10-fold concentration, is N-ethylmaleimide. The inhibition curves were the same for the two enzyme forms. Sodium Dodecyl Sulfate Gel Electrophoresis. Patterns for the two enzyme forms and their mixture are shown in Fig. 2. For ribonuclease H1 (gel A), the positions and the number of the faint intermediate bands are not constant; in some preparations only one band was interspersed at a different location. These bands are due presumably to contaminating proteins. In contrast, the pattern for ribonuclease H2 (gel B) was always the same. In the mixture (gel C), bands 1 of gels A and B migrated together, as did band 4 of gel A and band 3 of gel B. Their molecular weights have been estimated as 33,000 and 21,000,

_

_

Table 2. Nature of terminal nucleotides having free 3'-OH groups, produced by ribonucleases H1 and H2 from phage fl DNA-RNA hybrid* in presence of different activating metal ionst

Mg2+

% 3'-OH nucleotides produced Co2+ Mn2+

Base

H1

H2

H1

H2

H1

H2

A G C U

31.3 16.0 26.2 26.5

28.6 16.0 27.9 27.4

24.5 32.9 20.0 22.5

22.4 40.9 16.7 19.9

29.9 18.6 24.7 26.9

27.1 23.8 23.2 25.9

* The composition of the RNA moiety of the 1:1 phage fl DNA-RNA hybrid (3) was (in mole %): adenine (A), 33.6; guanine (G), 22.0; cytosine (C), 19.6; uracil (U), 24.8. t Each assay mixture (120,gl) contained: 0.05 M Tris-HCl (pH 8.0), 50,Mg of bovine serum albumin, 10 nmol of hybrid labeled in one of the four bases, together with the following additions. Activation by Mg2+: 0.1 M KCl, 25 mM MgCl2,4 ng of ribonuclease H1 or 5 ng of ribonuclease H2. Activation by Co2+: 0.35 M KC1, 20 mM CoCl2,6 ng of H1 or 8 ng of H2. Activation by Mn2+: 0.35 M KCl, 0.5 mM MnCl2, 40 ng of H1 or 50 ng of H2. (These conditions were first established with a hybrid labeled in all bases, so as to obtain the hydrolysis of 25-35% of the substrate.) After 15 min at 350 the reaction was stopped by the addition of 0.6 ml of uranyl acetate solution and the bases attacked during the enzymic hydrolysis were determined by assay B (4). The percentage figures reported in the table refer to % of all bases attacked.

A

B

C

FIG. 2. Electrophoresis of the two enzyme forms in presence

of sodium dodecyl sulfate. A, Ribonuclease H1; B, ribonuclease H2; C, mixture of both preparations. The bands, as referred to in the text, are numbered downward from the uppermost strong band designated as 1. Thus, gel A shows four bands and gel B three bands, of which band 2 cannot be seen clearly in the photograph.

4144

Biochemistry: Stavrianopoulos and Chargaff

respectively, whereas for both forms of ribonuclease H itself the molecular weight, from gel filtration, is 64,000 + 2000, in accordance with our previous estimate (1). It is, hence, likely that ribonuclease H consists of subunits, as was mentioned before (1). Contamination with Other Nucleases. Both enzyme preparations contained traces of a nuclease attacking nonhybridized RNA. The ratios of ribonuclease to ribonuclease H were 1:6000 for ribonuclease H1 and 1:4500 for ribonuclease H2. That this activity is not a property inherent in the hybridase has been shown by our previous procedure which yielded preparations entirely devoid of this activity (1). Nevertheless, the specimens described here have been used safely in many laboratories for the removal of poly(A) clusters from mRNA.

Concluding remarks As is shown here, freshly collected thymus from 4- to 6months-old calves yields two separable forms of ribonuclease H. Whether these should be considered as isoenzymes remains an open question. It is probably significant that the occurrence of ribonuclease H2 appears to be a function of the age of the donors. Preliminary experiments have shown that in 1-year old

Proc. Natl. Acad. Sci. USA 75 (1978)

animals only 8% of the total activity is attributable to fraction H2, whereas 85% resides in ribonuclease H1. This explains why in our previous study, performed with commercially available frozen tissue, only one form of the enzyme was encountered (1). It will also be noted that in the final specimens secured in the present work the specific activity was almost doubled. This work was supported by Research Grant CA 19289 from the National Institutes of Health, U.S. Public Health Service. 1. Stavrianopoulos, J. G. & Chargaff, E. (1973) Proc. Natl. Acad. Sci. USA 70, 1959-196. 2. Buesen, W. & Hausen, P. (1975) Eur. J. Biochem. 52, 179-190. 3. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) Proc. Natl. Acad. Sci. USA 69,2609-2613. 4. Stavrianopoulos, J. G. & Chargaff, E. (1976) Proc. Natl. Acad. Sci. USA 73, 1556-1558. 5. Polson, A., Potgieter, G. M., Largier, J. F., Mears, G. E. F. & Joubert, F. J. (1964) Biochim. Biophys. Acta 82,463-475. 6. Stein, H. & Hausen, P. (1969) Science 166,393-395. 7. Haberkern, C. & Cantoni, G. L. (1973) Biochemistry 12,23892395. 8. Stavrianopoulos, J. G., Gambino-Giuffrida, A. & Chargaff, E. (1976) Proc. Nati. Acad. Sci. USA 73, 1087-1091.

Simplified method for purification of ribonuclease H from calf thymus.

Proc. Natl. Acad. Sci. USA Vol. 75, No. 9, pp. 4140-4144, September 1978 Biochemistry Simplified method for purification of ribonuclease H from calf...
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