Proc. Nat. Acad. Sci. USAVol. 71, No. 11, pp. 4404-4408, November 1974

Terminal Deoxyribonucleotidyl Transferase in Human Leukemia(DNA polymerase/lymphocytes/marrow/thymus)

MARY SUE COLEMAN, JOHN J. HUTTON, PHILIP DE SIMONE, AND F. J. BOLLUM

Departments of Medicine and Biochemistry, University of Kentucky Medical Center and Veterans Administration Hospital,Lexington, Ky. 40506

Communicated by Elizabeth S. Russell, August 29, 1974

ABSTRACT Terminal deoxyribonucleotidyl transferase(EC 2.7.7.31; nucleoside triphosphate:DNA nucleotidyl.exotransferase) is usually found only in thymus, but hasbeen reported in leukemic cells from children with acutelymphoblastic leukemia. In an unusual adult patient withacute myelomonocytic leukemia, terminal transferasewas found at a level of 16 units per 108 bone marrow cellsand 14 units per 108 circulating leukocytes (1 unit = 1nmol of nucleotide per hr). This activity is comparable tothat found in normal thymus. Assays of transferase inmarrow and peripheral leukocytes from patients withtypical acute and chronic myelogenous leukemias gaveaverage values of 0.5 and 0.3 unit per 108 cells, respectively.Transferase activity is also found in normal bone marrowat about 0.07 unit per 10(8 cells. Terminal deoxyribonucleo-tidyl transferase in all samples of human marrow andperipheral blood had reaction characteristics, sedimen-tation, and chromatographic properties similar to thehomogeneous enzyme from calf thymus.

Terminal deoxyribonucleotidyl transferase (EC 2.7.7.31;nucleosidetriphosphate:DNA nucleotidylexotransferase) cat-alyzes the polymerization of deoxyribonucleotide triphos-phates, elongating polydeoxyribonucleotide chains withouttemplate instruction (1). This enzyme, originally found incalf thymus (2), has been detected only in thymus in a surveyof many species, and shows a marked increase during bovinefetal growth (3). The unique localization of the transferase isof uncertain biological significance, but there are speculationsthat it may play a role in conveying immunological specificity(or immunological diversity) to T and possibly B lympho-cytes during their differentiation (1, 3, 4). Demonstration ofdeoxyribonucleotidyl transferase activity in leukemic cells ofcertain children with acute lymphoblastic leukemia (5) andcell lines derived from patients with acute lymphoblasticleukemia (6) leads to the suggestion that malignant cells inacute lymphoblastic leukemia might arise from thymocytesor their precursors (5).

In this report an adult patient with acute leukemia, prob-ably myelomonocytic by morphological criteria, is shown tocontain deoxyribonucleotidyl transferase activity in leuko-cytes and bone marrow equal to levels usually seen in thymus.Because of the high level of transferase in cells from thispatient, comparison of its biochemical properties with thoseof the calf thymus enzyme is straightforward and the re-action characteristics of the two are practically identical.The transferase is also present in cells from patients with

Abbreviations: Transferase, terminal deoxyribonucleotidyl trans-

ferase; DNA polymerase-a and -,8, high- and low-molecular-weight DNA-dependent DNA polymerase, respectively; AML,acute myelogenous leukemia; MR cells, hematopoietic cells from

patient MR.

typical acute and chronic myelogenous leukemias, and verylow levels can be detected in normal adult bone marrow. Theenzyme in normal human cells and leukemic cells is alsoidentical to calf thymus transferase in all properties exam-ined. Quantitative estimates of transferase levels in normaland leukemic human hematopoietic cells are presented.

MATERIALS AND METHODS

Bone Marrow and Peripheral Blood. Protocols for thesestudies were approved by the University of Kentucky Com-mittee on Human Investigation. Diagnostic bone marrowaspirates and peripheral blood samples were collected in 5mM EDTA to prevent coagulation. Nucleated cells were sep-arated, counted, and frozen as described (7). The marrowaspirates from nonleukemic patients showed normal cellularmorphology both on smear and biopsy, except for severalwith mild iron deficiency.* Leukemic marrows and peripheralblood samples were taken from adult patients with untreatedacute myelogenous or myelomonocytic leukemia. In thesesamples, over 50% of the marrow cells were leukemic blasts.Specimens of leukemic cells from patient MR had unusualmorphological and biochemical properties. The clinical fea-tures of MR's illness are summarized as follows:MR was a 72-year-old Caucasian woman who had been

well all of her life. She reported to the clinic with a 2-monthhistory of malaise. Physical examination showed ecchy-moses, but no adenopathy or hepatosplenomegaly. The chestx-ray had no evidence of thymic enlargement. Initial labora-tory values were hemoglobin, 7.2 g/100 ml; leukocyte count,20,500/mm3 with a differential of 8 neutrophils, 8 stabs, 5metamyelocytes, 3 myelocytes, 2 promyelocytes, 12 lympho-cytes, 7 monocytes, 6 young monocytes, and 49 blasts; plate-lets, 11,000/mm3. A bone marrow aspirate showed about 95%leukemic blasts (Fig. 1)with a small amount of cytoplasm

* It is impossible to be certain that none of the normal marrows was"preleukemic." However, neither the morphology of the marrowsnor the subsequent clinical courses of the patients was compatiblewith leukemia. Bone marrow aspirates from normal individualsare rather difficult to obtain, and all such specimens provideonly a limited amount of material. In order to carry out theexperiment described in Results, we had to pool 16 bone marrow

aspirates. While the validity of this procedure can be questionedbecause one of the aspirates could have had an elevated level oftransferase, we feel that it is improbable. Analysis of individualhuman marrows and animal investigations lead us to believethat transferase level is very low in this tissue. Continued in-vestigation of the normal variability of nonleukemic specimenswill be necessary in order to validate the conclusions drawn fromthis single pooled specimen.

4404

Terminal Transferase in Leukemia 4405

BFIG. 1. Morphology of bone marrow cells from patient MR. Wright's stain. A, Field showing typical leukemic blasts. B, Field showing

monoblast typical of acute monocytic leukemia.

and some pseudopodia. Most nuclei contained 2-4 small nu-cleoli, and some nuclei were folded with monocytic features.An occasional typical monoblast was seen. Auer rods werenot found. About 10% of the blasts were peroxidase-positivewith large granules. Another 10% showed fine stippling whenstained with periodic acid-Schiff's reagent. The most likelydiagnosis was poorly differentiated, acute myelomonocyticleukemia (8), but acute lymphoblastic leukemia could not bedefinitively ruled out. The patient refused treatment in thehospital and received vincristine and prednisone at home.She died 5 weeks later with a leukocyte count of 210,000/mm3 and 80% blasts.

Preparation of Cell Extracts. All fractionation procedureswere done at 0-4°. Bone marrow or leukocytes were homog-enized in 0.25 M sucrose containing 50 mM Tris HCl (pH7.6), 25 mM KCl, 5 mM MgCl2 (sucrose TKM). The 100,000X g supernatant fractions were prepared essentially as de-scribed (7) and dialyzed against 25 mM Tris HCl (pH 8.0),0.5 M NaCI, 1 mM EDTA, and 1 mM 2-mercaptoethanol(buffer A). The dialyzed fraction is used directly for crudeextract assays. For further analysis a 0.25-ml aliquot waslayered onto a 5-ml 5-20% sucrose gradient in buffer A andcentrifuged for 18 hr at 100,000 X g in a Spinco SW 50.1rotor. Fractions (0.25 ml) were collected from the gradients bydisplacement from the bottom.

Enzyme Assays. Sources of nucleotides and of oligo- andpolydeoxyribonucleotides, and assays of DNA-dependentDNA polymerases-a and -fl have been described (7). Theterminal deoxyribonucleotidyl transferase reaction mixturecontained 0.20 M potassium cacodylate buffer (pH 7.0), 0.1mM [3H]dGTP (300-700 cpm/pmol), 0.02 mM d(pA)50, 1mM 2-mercaptoethanol, and 1 mM MgCl2. The rationale foruse of this assay and the use of sucrose gradients as aids in de-tecting transferase have been described earlier (9). Whenenzyme characteristics were performed, dNTP concentrationwas increased to 1 mM. Aliquots from the reaction mixturewere placed on Whatman GF/C glass fiber discs and processedfor acid-insoluble material. In experiments utilizing shorteroligodeoxyribonucleotide initiators, aliquots from the re-action mixture were placed on DE-81 discs, washed in 5%dibasic sodium phosphate, and dried with ethanol and ether

as for the GF/C discs. The dried DE-81 discs were incubatedovernight at 370 in Soluene 100 Tissue Solubilizer (PackardInstruments) before radioactivity was determined. Whenenzymes were assayed in the presence of N-ethylmaleimide,enzyme and inhibitor were incubated at 00 for at least 10 minbefore the addition of substrates. One unit of transferase orpolymerase activity equals 1 nmol of nucleotide polymerizedin 1 hr. Specific activities are expressed as units per 108 nu-cleated cells.

Other Methods. Isoelectric focusing was done on 7-cmcolumns containing 5% acrylamide and 1% pH 3-10 Am-pholines (LKB Instruments) with constant voltage. Hemo-globin and cytochrome c were used as pH markers. After pro-teins were focused (about 3 hr), the gels were sliced and theslices eluted with 25 mM Tris HCl (pH 8.0), containing 100mM KCl and 1 mM 2-mercaptoethanol.

Ion-exchange chromatography was done on DEAE-cellu-lose (DE-1 1) equilibrated with 50 mM Tris HCl (pH 8.0).Tissue extracts (100,000 X g supernatant fractions or ex-tracts concentrated by adsorption and elution from phospho-cellulose) were dialyzed against 50 mM Tris HCl (pH 8.0)and put onto 1 X 2-cm columns of DE-11. Under these condi-tions, DNA polymerase-, is not adsorbed, while transferaseand DNA polymerase-a bind (10). Transferase is eluted with50 mM KCl and DNA polymerase-a with 300 mM KCl in 50mM Tris HCl (pH 8.0).Antigen-antibody reaction mixtures contained 40 ,u of

fractions from sucrose gradients and 20 ,Al of rabbit anti-body directed against DNA polymerase-ca (20 mg of protein/ml), 0.1 M saline, or normal rabbit IgG (20 mg/ml). Theantigen-antibody mixtures were stored overnight at 00, andan aliquot was assayed for residual enzyme activity (11).

RESULTS

Transferase in Hematopoietic Cells from Patient MR. Dur-ing the last 2 years we have examined many human bonemarrow specimens for the presence of transferase as part of aquantitative survey of DNA polymerase activities in hemato-poietic cells (7). All of the specimens tested were from adults,and no patients with acute lymphoblastic leukemia were inthat series. Transferase was not detected at the level of sen-sitivity of the assay used at that time (80 cpm/pmol of dGTP,

Proc. Nat. Acad. Sci. USA 71 (1974)

4406 Medical Sciences: Coleman et al.

LEPER

PER

PERI

PERI

I r,

RATTHYMUS

LEUKEMICBONE

MARROW

-UKEMICIPHERAL

AML!IPHERAL

AMLBONE

MARROW

AMLIPHERAL

AMLIPHERAL

I 1

3 6 9 12 15 18nMOL of [3H]dGTP INCORPORATED/ 1 08 CELLS per HR

FIG. 2. Activities of transferase in various cells and tissues.Leukemic bone marrow and leukemic peripheral blood wereobtained from patient MR. AML peripheral blood and AMLbone marrow refer to samples from individual patients withtypical untreated adult acute myelogenous leukemia.

which will detect transferase at a level of about 1-2 units per108 cells). The unusual patient MR (see Materials and Meth-ods), had very high levels of transferase activity in bothmarrow and peripheral leukocyte samples. Transferase activ-ity was easily measured in crude extracts of bone marrow (16units per 108 cells) and peripheral blood (14 units). Thesevalues are thought to be reasonable estimates of transferaseactivity, even in the crude cell extracts, since the rate of in-corporation of dGTP onto d(pA)-0 was linear with proteinconcentration and time. The activity of transferase in MRcells was as high as the activity usually observed in rat thy-mocytes (15 units per 108 cells; see Fig. 2). It was similar tothe activity of DNA polymerase-a (61 units)t and muchhigher than DNA polymerase-#B (1.4 units per 108 cells),which sediments near transferase on sucrose gradients. Inleukocytes from patient MR, the transferase activity fell to0.3 units per 108 cells while she was being treated with pred-nisone and vincristine, although her peripheral blast countremained high.The enzyme activity from MR cells is readily demonstrated

to be similar to calf thymus transferase. The transferaseactivity on a sucrose gradient of extracts from MR cells isshown in Fig. 3. Homogeneous calf thymus transferase has amolecular weight of 32,000 and sediments to the same positionin separate gradients. Deoxyribocytidinetriphosphate poly-merization on d(pA)i- or d(pT)Io in the presence of Co2+ ex-hibits a rate about 3-fold greater than purinetriphosphatepolymerization. This unusual effect of Co+2 in dCTP poly-merization has also been described with the calf thymusenzyme (12). The initial rate of dGTP polymerization in the

Ia1)

0CoC]-J

LLJ

w

ODCL)

0

I-00j

cc

0

a.

0z

CL~0

4-0

0.

5 10 15T FRACTION NUMBER B

FIG. 3. Gradient analysis of terminal transferase. Extracts ofnucleated cells from MR and a patient with typical AML. DNApolymerase-a and -B (not shown) sediment in tubes 13 and 6,respectively. *, MR marrow; 0, MR peripheral blood; A, AMLperipheral blood.

presence of Mjg+2 is about equal when d(pG)5, d(pT)lo, ord(pA)ii serves as initiator. The transferase activity from MRcells is higher in the presence of Mg+2 than of Mn+2, contraryto other reports (5), but consistent with the properties of calfthymus transferase (12). Mn2+-dGTP polymerization withd(pG)5 appears to level off at 25 min. The problem of aggre-gation of dGTP products is well known and has been con-sidered in detail in an earlier publication (13). Polymeriza-tion of Mg+2-dATP was linear for 25 min and was comparablein rate to Mg+2-dGTP polymerization with either d(pT)lo ord(pA)-i. All of the characteristics of the initiator-addition re-actions observed in MR cell extracts are typical of homogene-ous calf thymus transferase (9), provided saturating concen-trations of initiator (0.01 mM oligonucleotide) and dNTP(1 mM) are present. They also serve to distinguish the trans-ferase activity in MR cells from DNA polymerase-,3.

Transferase from MR cells is not inhibited by antibody tocalf thymus DNA polymerase-a (Fig. 4), whereas humanpolymerases-a and -, are inhibited (ref. 11, and data notshown). When applied to DE-11 columns, transferase inextracts from MR cells was adsorbed and eluted with 50 mMKCl as expected (10). Transferase from MR cells also be-haves like the calf thymus enzyme on isoelectric focusing(pl1 7, Fig. 5) and is easily distinguished from DNA poly-merase-,B (pl > 9; see refs. 14 and 15). These results demon-strate that the transferase activity in MR cells is similar tocalf thymus transferase and is not a minor reaction catalyzedby DNA polymerase-f3.

t DNA polymerase units are expressed as total nucleotide poly-merized, i.e., 3.4 X radioactive nucleotide for activated calfthymus DNA template.

Proc. Nat. Acad. Sci. USA 71 (1974)

Terminal Transferase in Leukemia 4407

0

0

a. Saline00 5 -

z

0_sl A/ntiO0(

Z 3 _0

0

2 -

5 10 20 25TIME (MINUTES)

FIG. 4. Effect of antibody to calf thymus DNA polymerase-aon transferase. The enzyme used was a sucrose gradient fractionfrom an extract of MR leukemic cells. The reaction mixturescontained d(pA)50 and [3H]dGTP. 0, Transferase + saline; 0,

transferase + antibody against DNA polymerase-a.

Transferase in Acute Myelogenous Leukemia (AML). WithdGTP of high specific activity (300-700 cpm/pmol), transfer-ase could also be detected in extracts of cells from the pe-ripheral blood and bone marrow of adult patients with typicaluntreated AML. Fig. 3 illustrates the sedimentation rate oftransferase activity in an AML extract with 0.9 unit per 108cells. Transferase from MR cells, AML cells, and calf thymussediment to the same position in the sucrose gradient. Ex-tracts of eight samples of marrow from patients with AMLwere sedimented separately on sucrose gradients, and transfer-ase was assayed. All samples had transferase activity, rangingfrom 0.3 to 1.0 unit per 108 cells, and in all cases transferasewas found at the appropriate position in the gradient.

It is particularly important to distinguish unequivocallybetween transferase and DNA polymerase-,B in samples withlow activity since the two activity levels are approximatelythe same and they sediment together on a sucrose gradient.Unlike DNA polymerase-,B, transferase activity in AML cellswas not inhibited by antibody to calf thymus DNA poly-merase-a. As a further characterization of the enzyme in AMLextracts, we examined behavior of the activity on DE-11columns since DNA polymerase-fl is readily separated fromtransferase on such columns (10). Five separate extracts frommarrows of AML patients were chromatographed on DE-11.Four of these were obtained from different individual donors,while the fifth was a pooled sample from three patients. Inall cases DNA polymerase-, activity passed through the

FIG. 5. Isoeectricfocusin, pH 3transferaseZ 80

U-positions ofhmgoin(b n ctcrm Hb CYTco

-

0

0.-:0

5 O 1 2 5 3 3 0 4

marked.

column, whereas transferase activity was adsorbed and elutedat 50 mM KCl. From the activity of transferase in eluatesfrom the five different AML samples, we calculate a specificactivity of 0.9-1.1 units per 108 cells. We have also studiedextracts of peripheral leukocytes from one patient withchronic myelogenous leukemia, graciously supplied by Dr.Robert Gallo. A transferase activity of 0.3 unit per 108 cellswas found, comparable to that seen in AML cells.

Transferase in Normal Marrow and Lymphvocytes. Sincetransferase activity could be demonstrated in AML cells, wereasoned that transferase might be detected in normal mar-row, possibly even in normal lymphocytes, if appropriateconcentrating procedures were used. Sixteen specimens ofnormal marrow were pooled to obtain about 3 X 109 cells.The soluble supernatant fraction was prepared, and the de-oxyribonucleotide polymerizing enzymes were adsorbed to aphosphocellulose column. Activity was eluted with a high saltwash (16), dialyzed against 50 mM Tris .HCl (pH 8.0), andthen adsorbed onto a DE-11 column. Upon elution with 50mM KCl, a peak of transferase activity was observed. Fromthis experiment the activity of transferase in normal marrowis estimated at 0.07 unit per 108 cells.

Concentrated extracts of large numbers of lymphocytespurified from normal peripheral blood (1.7 X 109 cells), phyto-hemagglutinin-stimulated normal lymphocytes (1.2 X 108cells), and from the blood of a patient with chronic lymphocyticleukemia (1010 cells) were examined onsucrosedensitygradientsand by chromatography on DE-11 columns. It is estimatedthat these cells contain less than 0.01 unit of transferaseactivity per 108 cells since no activity was detected.

Proc. Nat. Acad. Sci. USA 71 (1974)

4408 Medical Sciences: Coleman et al.

Inhibitor Studies. Studies with inhibitors also serve todistinguish transferase from DNA polymerases-a and -0.Transferase from marrow of MR, patients with AML, andnonleukemic patients was inhibited 60% by 10% ethanol andinhibited completely by a combination of 10% ethanol and10 mM N-ethylmaleimide. DNA polymerase-0 was not in-hibited under these conditions. DNA polymerase-a is com-pletely inhibited by 10 mM N-ethylmaleimide.

DISCUSSION

The results of this investigation demonstrate that terminaldeoxyribonucleotidyl transferase occurs in "normal" bonemarrow as well as in certain leukemic states. It is not uniqueto thymus nor is it unique to acute lymphoblastic leukemiccells, occurring in patients with chronic myelogenous leu-kemia and AML as well. Unusually high levels were found incells of an adult patient with poorly differentiated acute mye-lomonocytic leukemia. The quantitative results show thatoccurrence of transferase outside of thymus is indeed anunusual event, and a readily detectable level in nonthymictissue is probably indicative of pathology.The trace of transferase activity found in "normal" bone

marrow* may be of considerable biological significance. Dataobtained in animal experiments have raised the concept ofpluripotential stem cells which differentiate into separateprogenitor cells for lymphoid and myeloid lines (17). One ofthe striking features of the peripheral blood and bone marrowof patient MR is the extreme morphologic variation. Cellsresembling myeloblasts, lymphoblasts, monoblasts, mono-cytes, and granulocytes of various stages of maturity werepresent. One could imagine that malignant transformationhad occurred in a pluripotential stem or early progenitor cellwith production of multiple types of more differentiated cellsin both the myeloid and lymphoid lines. Although transferaseis normally present in high concentration only in thymus, itspresence in hematopoietic cells of patient MR and childrenwith acute lymphoblastic leukemia (4, 5) suggests that lym-phoid progenitor cells from bone marrow as well as thymo-cytes may contain transferase. The major reservoir of lym-phoid progenitor cells in adults is thought to be bone marrow(17), an I such cells are probably rare. From cell separationstudies on rat thymus we estimate that between 10 and 50%of the cells contain transferase (18). Since normal bone mar-row shows only about 1/350th the level of thymus, we esti-mate that only 0.03-0.15% of normal bone marrow cells willhave this activity. If one accepts the assumptions implicit inthis calculation-that is, that the cellular content of enzymein bone marrow cells is the same as for thymus and is re-stricted to a specific class of cell-then the bone marrow cellcontaining this activity will be rare indeed.Now that transferase activity is found in more locations of

biological interest,t the urge to speculate on its possible

t Since submission of this manuscript, B. I. S. Srivastava [CancerResearch (1974) 34, 10151 and C. Penit, A. Paraf, and F. Chape-ville [Nature (1974) 249, 755] have reported terminal trans-ferase activity in nonthymic cells. We do not feel it is appropriateto make a critical comparison at this time.

biological role becomes almost irrepressible. But for the mo-ment, it is sufficient to restate that we think the enzyme hassomething to do with immunological diversity (1, 3, 4). Weare still concerned about the accuracy of measurement of theenzyme activity in samples of low activity. That is, is theenzyme really low or absent, or is it masked in some way inthese samples? Continued investigation will provide theanswer to that and other questions on the biological sig-nificance of terminal deoxyribonucleotidyl transferase.

Note Added in Proof. Since submission of this manuscript, 9individual human bone marrows from nonleukemic patients havebeen analyzed. Five of these gave definitely positive results fortransferase in the crude extract. Analysis of normal rabbit bonemarrow and normal rat bone marrow also gives clearly positiveresponses. All of these results substantiate the claim that lowlevels of transferase can be detected in normal bone marrow.

Dr. Lucy Chang provided rabbit antibody to DNA poly-merase-a from calf thymus, and homogeneous DNA polymerase-,3from calf thymus. She also suggested the use of ethanol and N-ethylmaleimide as a differential test for transferase and DNApolymerase-3. This research is supported in part by Grants AM16013 and CA 08487 from the National Institutes of Health andby Grant KTRB 064 from the University of Kentucky Tobaccoand Health Research Institute.

1. Bollum, F. J. (1974) in The Enzymes, ed. Boyer, P. D.(Academic Press, New York), pp. 145- 171.

2. Bollum, F. J. (1960) J. Biol. Chem. 235, PC18.3. Chang, L. M. S. (1971) Biochem. Biophys. Res. Commun.

44, 124-131.4. Baltimore, D. (1974) Nature 248, 409- 411.5. McCaffrey, R., Smoler, D. F. & Baltimore, D. (1973) Proc.

Nat. Acad. Sci. USA 70, 521- 525.6. Srivastava, B. I. S. & Minowada, J. (1973) Biochem.

Biophys. Res. Commun. 51, 529- 535.7. Coleman, M. S., Hutton, J. J. & Bollum, F. J. (1974) Blood

44, 19-32.8. Hayhoe, F. G. (1969) Seminars in Hematology 6, 261-270.9. Chang, L. M. S. & Bollum, F. J. (1971) J. Biol. Chem. 246,

909-916.10. Bollum, F. J., Chang, L. M. S., Dorson, J. W. & Tsiapalis,

C. M. (1974) in Methods in Enzymology, eds. Grossman, L.& Moldave, K. (Academic Press, New York), Vol. XXIX,pp. 70-81.

11. Chang, L. M. S. & Bollum, F. J. (1972) Science 175, 1116-1117.

12. Kato, K., Gongalves, J. M., Houts, G. E. & Bollum, F. J.(1967) J. Biol. Chem. 242, 2780-2789.

13. Lefler, C. F. & Bollum, F. J. (1969) J. Biol. Chem. 244,594- 601.

14. Chang, L. M. S. (1974) in Methods in Enzymology, eds.Grossman, L. & Moldave, K. (Academic Press, New York),Vol. XXIX, pp 87-88.

15. Sedwick, W. D., Wang, R. S. F. & Korn, D. (1974) inMethods in Enzymology, eds. Grossman, L. & Moldave, K.(Academic Press, New York) Vol. XXIX, pp. 89-102.

16. Yoneda, M. & Bollum, F. J. (1965) J. Biol. Chem. 240,3385-3391.

17. Metcalf, D. & Moore, M. A. S. (1971) in HaematopoieticCells (North Holland Publishing Co., Amsterdam), pp.264-271.

18. Coleman, M. S., Hutton, J. J. & Bollum, F. J. (1974)Biochem. Biophys. Res. Commun. 58, 1104-1109.

Proc. Nat. Acad. Sci. USA 71 (1974)