Defective DNA synthesis in megaloblastic anaemia. Studies employing velocity sedimentation in alkaline sucrose gradients

46

Biochimica et Biophysica Acta, 563 (1979) 46--58

© Elsevier/North-HollandBiomedicalPress

BBA 99459 DEFECTIVE DNA SYNTHESIS IN MEGALOBLASTIC ANAEMIA STUDIES EMPLOYING VELOCITY SEDIMENTATION IN ALKALINE SUCROSE GRADIENTS R. G I T E N D R A W I C K R E M A S I N G H E

and A. V I C T O R H O F F B R A N D

Department of Haematology, Royal Free Hospital,Pond Street,London, N W 3 2 Q G (U.K.) (Received January 2nd, 1979) Key words: DNA replication; Megaloblastic anemia; Vitamin B-12 deficiency; Folate deficiency; (Lymphocyte)

Summary 1. Autoradiographic experiments revealed that the average size of the replicating unit (replicon) in human phytohaemagglutunin-stimulated lymphocytes is 45 (_+ 1.3)/~m. 2. A 5 min pulse of [3H]thymidine labelled DNA chains of approximately 40 S (15 pro) in control lymphocytes as revealed by velocity sedimentation in alkaline sucrose density gradients. Upon chasing in the absence of [3H]thymidine the labelled DNA increased in size. By 6 h the bulk of the label co-sedimented with full-sized chromosomal DNA. 3. In untreated lymphocytes from patients with megaloblastic anaemia due to vitamin B-12 or folate deficiency or lymphocytes treated with methotrexate (10 -5 M) or hydroxyurea (5 • 10 -' M) the increase in size of pulse.labelled DNA was slower than in control cells. 4. The block in maturation of pulse-labelled DNA to bulk DNA was not permanent. At 24 h of chase 75--80% of the pulse-label in both control and megaloblastic lymphocytes co-sedimented with bulk DNA. 5. We conclude that the lesions seen in DNA synthesis in megaloblastic anaemia due to folate or vitamin B-12 deficiencies occur through impaired biosynthesis of nucleotide precursors of DNA. Possible explanations of why the defects in DNA synthesis cause altered morphology of proliferating cells in megaloblastic anaemia are suggested.

Introduction

Replication of chromosomal DNA of mammalian cells occurs in tandemly arranged units (replicons) 15 to 100 pm in length [1,2]. Two replication forks

47 initiate synthesis at the origin of each replicon and travel in opposite directions. When adjacent units have completed replication, the daughter DNA strands in each are joined together giving rise ultimately to chromosome-sized daughter duplexes. This sequence is reflected in the results of studies on DNA replication in Chinese hamster [3], Erlich ascites [4] and mouse L [5] cells using velocity sedimentation in alkaline sucrose gradients. A brief pulse (about 0.5 min) of [3H]thymidine labelled short DNA fragments of 4 S or less (Okazaki pieces). These fragments can be chased into intermediate-sized DNA (about 25 S) corresponding to growing replicon-sized DNA strands. These strands increase in length to about 70 S (presumably by growth within replicons and by joining together of completed units.) These labelled intermediates are finally chased into material co-sedimenting with bulk DNA. Megaloblastic anaemia, whatever the cause clinically, is characterised by lesions at the level of DNA replication [6,7]. This lesion is most obvious in the rapidly proliferating erythroid cells in bone marrow, resulting in the accumulation of abnormal erythrocyte precursors (megaloblasts) which show decondensed chromatin, large size and frequent chromosomal breakage at mitosis. Megaloblastic anaemia is caused by deficiency of Vitamin B-12 or folate or by treatment of patients by drugs that interfere with deoxyribonucleotide biosynthesis (e.g. hydroxyurea, methotrexate) or with DNA replication directly (e.g. cytosine arabinoside). Lymphocytes from patients with megaloblastic anaemia when cultured in the presence of phytohaemagglutunin show the characteristic morphological and biochemical abnormalities of megaloblasts [8,9]. These defects can be corrected by the addition to the culture medium of folinic acid and Vitamin B-12. Preliminary work carried out in this laboratory showed that DNA labelled for 1 h in lymphocytes from patients with megaloblastic anaemia sedimented more slowly in alkali compared to similarly labelled DNA from lymphocytes from a normal subject [7]. The studies described in this communication were carried out to define the lesion in DNA replication in megaloblastic anaemia in greater detail. Materials and Methods

Reagents. [3H]thymidine (48 Ci/mmol) was purchased from the Radiochemical Centre, Amersham, U.K. Medium 199 and phytohaemagglutunin were supplied by Wellcome Laboratories Ltd., Beckenham, Kent. All other reagents were of analytical grade. AR10 autoradiographic stripping film and D-19 developer were purchased from Kodak Ltd., Hemel Hempstead, Herts. Lymphocyte culture. Venous blood was obtained from normal subjects or from patients with megaloblastic anaemia (haemoglobin less than 10.0 g/dl) due to nutritional folate deficiency or to Vitamin B-12 deficiency due to pernicious anaemia. Lymphocytes were prepared from heparinized peripheral blood by sedimentation through a Triosill-Ficol gradient [10]. The cells were cultured in TC 199 medium supplemented with 20% autologous serum and 10 pl/ml phytohaemagglutunin. 3-ml aliquots of cultures were maintained in 5-ml bijou bottles at 37°C for 72 h prior to pulse-labelling. Lymphocytes from five

48 patients with megaloblastic anaemia were cultured in medium lacking thymine and folic acid. Three patients had nutritional folate deficiency and two had Vitamin B-12 deficiency due to pernicious anaemia. Folinic acid (10 ~g/ml) and Vitamin B-12 (3 pg/ml) were added at zero time to half of these cultures to provide controls displaying normal morphological and biochemical characteristics [8,9]. Pulse-chase procedures. L y m p h o c y t e s were concentrated to 35 • 106 cells/ml b y low speed centrifugation and resuspension. 0.5 ml-aliquots of this suspension were pulse-labelled for 5 min with [3H]thymidine (200 uCi/ml, 48 Ci/mmol). In experiments on cells from patients with megaloblastic anaemia or those treated with methotrexate, the pulse was terminated by dilution of the culture to 10 ml with used medium, low speed centrifugation, washing of the cells, and finally, resuspension in 6 ml of the appropriate medium without [3H]thymidine. Pulse-labelling of cells treated with h y d r o x y u r e a was terminated b y dilution with 6 ml medium containing 0.1 mM unlabelled thymidine and 0.5 mM hydroxyurea. 1-ml aliquots were taken at the time of termination o f the pulse and at later times and plunged into 4 ml absolute alcohol at

--20°C. The cells were collected by centrifugation at 700 × g for 5 min at 2°C, washed once in cold phosphate-buffered saline and taken up in the same to a b o u t l 0 T cells/ml. Alkaline sucrose density gradient sedimentation. 4.8-ml 5--20% sucrose gradients were prepared in siliconized 5 ml polyallomer centrifuge tubes. The gradient contained 0.1 N NaOH, 0.9 M NaC1 and 1 mM EDTA. 50 ~1 of a lysis solution (0.5 N NaOH, 10 mM Na2 EDTA) was layered on each gradient and 50 pl of the labelled cell suspension pipetted gently above this. A b o u t 5 • 10 s cells were loaded on each gradient, corresponding to a b o u t 2.5 ug of DNA. Gradients were kept for 16 h in the dark at 4°C to permit cell lysis with minimal shearing of DNA [5]. Prior to centrifugation a 0.2 ml cushion of 70% sucrose was injected beneath each gradient and the gradients centrifuged at 160 000 × g for 45 to 70 min at 16°C in the 6 × 5.5 ml swing-out rotor of an M.S.E. superspeed 75 centrifuge. 0.2 ml fractions were collected from the b o t t o m of each tube. Acid precipitable radioactivity was determined in aliquots (0.1--0.15 ml) of each fraction by the method of Bollum [11]. Scintillation counting was carried o u t in a Wallac 1210 liquid scintillation counter using a toluene-based scintillation cocktail. Where appropriate, correction was made for overlap of 14C into the 3H channel. Recoveries of loaded radioactivity were greater than 90%. Under these condition, a bulk DNA marker (prepared by labelling lymphocytes for 24 h with 0.5 gCi/ml [14C]thymidine) sedimented as an aggregate onto the 70% sucrose cushion. Typically, more than 90% o f the 14C-label was recovered in the b o t t o m fraction. To eliminate the possibility that low molecular weight material might be trapped b y the bulk D N A aggregate, or that the sedimentation position of this material may be altered b y the large amount o f DNA present the following experiment was performed. 0.5 pg o f a sample o f purified [3H]DNA (6500 cpm/pg) was sheared b y three passages through a 19G syringe needle and loaded onto an alkaline sucrose gradient. A replicate sample was loaded onto a parallel gradient together with 5 • l 0 s cells labelled for 24 h

49 with [14C]thymidine. Following centrifugation at 160 000 × g for 2 h at 16°C, the peaks of 3H radioactivity in the two gradients were superimposable, although the ~4C-label in the second gradient had sedimented onto the cushion. Estimation of sizes of intermediates. Gradients were calibrated by the manual calculation method of Steensgaard and Funding [12]. The accuracy of calibration was assessed using a [3H]adenovirus-5 DNA marker (35 S). Molecular weights were calculated from sedimentation coefficients using the Studier equation [13] and lengths of labelled intermediates obtained from these values. DNA fibre autoradiography. Normal lymphocytes cultured for 72 h were labelled for 30 min with [3H]thymidine at a spec. act. of 48 Ci/mmol and for a further 20 min at 8 Ci/mmol. The labelled cells were washed with cold saline and lysed on microsope slides as described by Hand and Tamm [14]. After fixing in 5% trichloroacetic acid and ethanol, the slides were dried and covered with Kodak AR10 stripping film. Slides were developed (Kodak D-19, 20 min) after exposure at 4°C for 10 weeks. The grain tracks were projected onto paper using a GS microscope fitted with a projection head. Grain tracks were traced with a pencil and centre to centre distances of bidirectional replicating units measured as described [ 14]. Calibration was achieved by measuring the lengths of the markings of a projected Neubauer haemocytometer. Results

DNA fibre autoradiography In order to facilitate the interpretation of the sedimentation studies described below, we determined the average size of the replicating unit in human lymphocytes by DNA fibre autoradiography. Fig. 1 shows the distribution of centre to centre distances of 251 segments of replicating DNA visualised by autoradiography. The lengths in the main peak vary from 20 to 80 pm with a mean value of 45 (+1.3) ~m. A small second peak with a mean size of 90 ~m is also discernible. The data suggests that the size of the average replicating unit is 45 pm, and that the occasional failure of a small number of replicons to

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50 initiate DNA synthesis generates the second peak at 90 pm. The value obtained by us for the average size of a replicon is within the range observed in mammalian cells by several other authors (reviewed by Edenberg and Huberman [15]). Sedimentation studies in megaloblastic anaemia Fig. 2a shows typical alkaline sedimentation profiles of pulse-labelled DNA of untreated and vitamin-treated lymphocytes from a patient with megaloblastic anaemia. The increased incorporation of [3H]thymidine in the untreated as compared to the treated cells (see legend to Figs. 2 and 3) is due to the reduced d e novo biosynthesis of dTTP in the untreated cells [9]. A 5 rain pulse-labelling of the treated cells labeled a broad peak centred at 45 S (22 pm) with a shoulder at higher S values. On chasing in the absence of [3H]thymidine for 25 min the size of the peak increased to 50 S (Fig. 2b).

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51 By 1 h of chase the bulk of the label sedimented as a broad peak centred at 85 S (Fig. 2c). On continued incubation this peak diminished in size (Fig. 2d) and was chased almost completely into material co-sedimenting with bulk DNA by 3 h (Fig. 2e). This general pattern was observed using lymphocytes from

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Alkaline sedimentation profiles of DIqA pulse-labelled for 5 rain with [3H]thymidine and chased 1 ( b ) 3 ( c ) 6 a n d ( d ) 2 4 h. L y m p h o e y t e s w e r e f r o m a p a t l e n t w i t h m e ~ l o b l a s t l e a n a e m i a c u l t u r e d presence (o) or absence (e) of added vitamins. About 8500 and 28 000 epm, respectively, were o n e a c h g r a d i e n t i n t h e t w o series. C e n t r i f u g a t i o n w a s f o r 6 0 m i n .

52 different donors, although some variability was observed in the timing of events (Figs. 3, 4, and 5). In the untreated cells, a 5 min pulse of [3H]thymidine labelled a broad peak centred at 35 S (12 ~m). The rate of increase in size of the pulse-labelled DNA during the subsequent chase and the appearance of label in material co-sedimenting with bulk DNA was markedly slower in these cells (Figs. 2b--e). Fig. 3 shows the results of a pulse-chase experiment carried out over a longer

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53 period. In this experiment a block in maturation of the labelled intermediates at 70 S was evident in the untreated cells as compared to the treated cells (Figs. 3a and b). However, the block was not permanent and by 6 h the pulselabel had started to enter the bulk DNA (Fig. 3c). By 24 h the sedimentation profiles of the treated and untreated cells were almost identical, except that a small peak of low molecular weight material at about 23 S was more prominent in the untreated cells (Fig. 2d). The DNA of partially purified mitochondria prepared from lymphocytes labelled for 20 h with [3H]thymidine was found to sediment at 24 S in alkaline sucrose gradients (Wickremasinghe, R.G., unpublished observations). It is likely that the 23-S peaks of Fig. 3d represent mitochondrial DNA. In the experiment described in Figs. 2 and 3 there was a marked lag in the transfer of pulse-label to material co-sedimenting with bulk DNA (Table I, columns 1 and 2). Columns 3, 4 and 5 summarise the results of experiments

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Fig. 5. A l k a l i n e s e d i m e n t a t i o n p r o f i l e s of D N A pu]se-labeUed for 5 m i n w i t h [ 3 H ] t h y m i d i n e (a) a n d c h a s e d for (b) 2 5 (e) 6 0 (d) 1 2 0 a n d (e) 1 8 0 rain. L y m p h o e y t e s f r o m a n o r m a l s u b j e c t p r e i n e u b a t e d for I h i n t h e a b s e n c e (o) o r p r e s e n c e ( e ) of 0 . 5 M h y d r o x y u r e a . I n h i b i t i o n of [ 3 H ] t h y m i d i n e i n e o r p o r a t i o n w a s 92%. A b o u t 75 0 0 0 a n d 6 7 0 0 cprn, r e s p e c t i v e l y , w e r e l o a d e d o n e a e h g r a d i e n t in t h e t w o series.

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54 TABLE I ENTRY OF PULSE LABEL INTO BULK DNA IN LYMPHOCYTES FROM PATIENTS WITH MEGALOBLASTIC ANAEMIA The percentage of [ 3 H ] t h y m i d i n e pulse-labelled D N A co-scdimenting with [ 1 4 C ] t h y m i d i n e uniformlyl a b e l l e d D N A in a l k a l i n e s u c r o s e g r a d i e n t s in five s e p a r a t e e x p e r i m e n t s w a s c o m p u t e d . (--) in t h e a b s e n c e o r ( + ) in t h e p r e s e n c e o f a d d e d v i t a m i n B 12 a n d f o l i n i e a c i d . C o l u m n s 1 a n d 2 r e f e r t o t h e e x p e r i m e n t s d e s c r i b e d in Figs. 2 a n d 3, r e s p e c t i v e l y . C a s e s 1, 3 a n d 5 w e r e d i a g n o s e d as p e r n i c i o u s a n a e m i a a n d cases 2 a n d 4 as n u t r i t i o n a l f o l a t e d e f i c i e n c y . All c a s e s h a d m e a n c o r p u s c u l a r v o l u m e s in e x c e s s o f 1 1 5 fl a n d f l o r i d e r n e g a l o b l a s t i c m a r r o w s , a n d h a e m o g l o b i n less t h a n 9 . 0 g / d l . Chase time

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carried out on lymphocytes from three other patients. These experiments also support the view that maturation of pulse-labeUed DNA intermediates into chromosome-sized strands is delayed in untreated cells from patients with megaloblastic anaemia. It seemed possible that the difference seen by us between the untreated and vitamin-treated cells were artefacts resulting from culturing o f lymphocytes in medium lacking thymine and folic acid. However, when similar experiments were performed on cells from a normal individual, no differences were observed between cells grown in the presence or the absence of added vitamins (data not shown). Therefore, the lesions described in the untreated cells were the result of an intrinsic defect in the cells themselves or in the autologous serum used in the cultures. S e d i m e n t a t i o n studies on l y m p h o c y t e s treated with m et hot rexat e or h y d r o x y . urea

For comparison, similar studies were made using cells treated with the cytotoxic drugs methotrexate and hydroxyurea. Both these drugs are known to cause megaloblastic anaemia in vivo [7]. Treatment of lymphocytes from a normal donor with 10 -s M methotrexate (Fig. 4) or 0.5 mM hydroxyurea (Fig. 5) produced effects similar to those seen in untreated !ymphocytes from patients with megaloblastic anaemia. In both these experiments the conversion of DNA strands labelled by a 5 min pulse to material co-sedimenting with bulk DNA was markedly retarded in the drug-treated cells as compared to the controis.

Discmmion The results of this study demonstrate retarded conversion of pulse-labelled DNA to full-sized DNA strands in megaloblastic anaemia.

55 The procedure used by us to study the DNA replication in lymphocytes has minimised the possibility of artefacts arising due to the shearing of DNA [15,16] or to trapping of labelled intermediates by the aggregate of bulk DNA (see Materials and Methods). Furthermore, the observation of regular arrays of labelled segments in autoradiographs of DNA from both treated and untreated lymphocytes (Wickremasinghe, R.G. and Hoffbrand, A.V., unpublished data) suggest that the bulk of incorporation of radioactive precursor is due to semiconservative replication rather than repair synthesis. The conventional test for repair synthesis (e.g. see Ref. 17) could not be applied due to the possibility that the required prelabelling with Bromodeoxyuridine correct defects caused by disturbed nucleotide biosynthesis. Normal lymphocytes or vitamin-treated lymphocytes from patients with megaloblastic anaemia labelled a broad peak of DNA chains centred at about 40 S (15 ~m) on exposure to a 5 rain pulse of [3H]thymidine (Figs. 2a, 4a and 5a). A shoulder at higher S values is also present. Fridland [18] has also observed that an 8 min pulse-labelling of human lymphoblasts labelled a broad peak at 38 S with a prominent shoulder. The heterogenous sedimentation behaviour of pulse-labelled DNA in this system can be attributed to the nonuniformity of replicon size in cultured lymphocytes (Fig. 1) and to the fact that replicons at varying stages of completion of DNA synthesis are labelled by the pulse. Nevertheless the size range of the peak labelled initially by the pulse, taken together with the size distribution of replicating units in lymphocytes (mean size 45 Urn, see Fig. 1) suggests that the former represents growing DNA strands within individual replicons. It follows that increase in size of the labelled material during a chase must involve continuous growth of these strands due to replication fork movement until the nascent molecules attain the size of a replicon. Further increase in size must be due to the fusion of nascent strands synthesized by contiguous replicating units. In vitamin-treated lymphocytes from patients with megaloblastic anaemia the size of the pulse-labelled peak increases on chasing, until a t 3 h the major proportion of the label sediments with bulk DNA (Fig. 2). A similar pattern is exhibited by lymphocytes from normal donors, although there is some variability in details of the gradient profiles and in the timing of events (Figs. 4 and 5). The increase in size of pulse-labelled DNA and its conversion to full-length strands is markedly slower in untreated megaloblastic lymphocytes (Fig. 2). However, due to the heterogenous sedimentation behaviour of the pulselabelled DNA it is not possible to say with any certainty if either replication fork movement or the joining of completed replicons or both processes are retarded in the untreated cells. However, the block in DNA maturation was n o t a permanent one, and continued incubation resulted in the maturation of the majority of the pulse-labelled fragments to material co-sedimenting with bulk DNA by 24 h of chase (Figs. 3c and d). A small peak of material sedimenting at 23 S persisted at 24 h in the untreated lymphocytes (10% of total recovered 3H cpm). A smaller peak (5.6% of total recovered 3H cpm) at the same position was evident in vitamin-treated lymphocytes (Fig. 3d). Since mitochondrial DNA of lymphocytes sediments at 24 S, the peak at low S values in cells chased for 24 h probably represents mitochondrial DNA. The delay in maturation of pulse-labelled DNA into bulk DNA was found to

56 be typical of patients with megaloblastic anaemia. In five experiments using lymphocytes from five different patients, the rate of appearance of pulse-label in bulk DNA was consistently lower in the untreated cells as compared to corresponding vitamin-treated controls (Table I). This difference was only seen in untreated cells from patients with megaloblastic anaemia. Lymphocytes from normal subjects, when cultured in the same manner, showed identical patterns of DNA synthesis whether grown in the presence or absence of added vitamins. Pulse-chase studies carried out on lymphocytes treated with either methotrexate or hydroxyurea revealed lesions similar to those exhibited by megaloblastic lymphocytes. Cells treated with either drug showed a retarded increase in size of DNA chains labelled by a brief pulse of [3H]thymidine (Figs. 4 and 5). The effects in drug-treated cells were more pronounced than in the untreated megaloblastic lymphocytes. This difference can be attributed to the fact that these drugs cause a rapid and drastic fall in certain nucleotide pools whereas the effects of chronic vitamin deficiency on pools are less marked [7,19]. The accumulation of labelled DNA in 40--80 S size range in cells treated with methotrexate [17] or hydroxyurea [20] has been observed previously. In contrast, other authors have described the hydroxyurea-induced accumulation of Okazaki pieces (about 4 S} during the replication of SV40 [21], polyoma virus [22] and mouse L cell [23] DNA. The discrepancy between sets of observations may be due to the degree of inhibition caused by the drug, since in cases where Okazaki pieces have been seen to accumulate, inhibition of [3H]thymidine incorporation has been in excess of 99% [21--23]. In the experiment described in Fig. 5 of this paper, inhibition was only 90%. Both the above-mentioned drugs interfere with the biosynthesis of deoxyribonucleotide precursors of DNA, although by quite distinct mechanisms. Methotrexate inhibits the enzyme dihydrofolate reductase, thereby inhibiting indirectly the folate
57 joining together of nascent DNA strands of contiguous replicons could be directly affected in the untreated cells. Indeed, recent evidence suggests that termination of SV40 DNA synthesis is a rate-determining step in the production of completed daughter chromosomes [29]. A second alternative that cannot be ruled out is that the failure of some replicons to initiate DNA synthesis may result in the retarded joining of the nascent strands synthesised within neighbouring replicons. Fridland and Brent have suggested that further increase in size of 82 S fragments accumulating in methotrexate-treated human lymphoblasts may be dependent on the synthesis of new 82 S fragments [17]. We are at present attempting to evaluate the above alternative by direct measurement of rates of replication fork movement and by DNA fibre autoradiography. Preliminary autoradiographic studies indicate no differences in replicon size distribution between treated and untreated cells (Wickremasinghe, R.G., unpublished data). The abnormal erythrocyte precursors (megaloblasts) characteristic of megaloblastic anaemia show peculiar cytological features such as decondensed chromatin, chromosome breakage at mitosis and an abnormally large size [7]. The connection between these abnormalities and the defects in DNA replication reported here remain to be elucidated. Setterfield, et al have observed that two different temperature-sensitive strains of mouse L cells suffer chromatin decondensation at the restrictive temperature [30]. In one of these strains the de novo synthesis of histone is repressed coordinately with DNA synthesis although pre-formed chromatin is fully conserved [31]. There is evidence that transient cycles of decondensation and condensation of chromatin related to DNA synthesis occur during the S phase of normal eukaryotic cells [32,33]. Setterfield, et al have proposed that the recondensation of chromatin decondensed for DNA replication is dependent upon normal termination of replication by groups of replicons [30]. If this interpretation is correct, the abnormal organisation of chromatin in megaloblasts could be seen as resulting from the lag in maturation of replicating intermediates to bulk DNA. Mitchison [34] has discussed the possibility that inhibition of the DNA replication cycle may result in an uncoupling of the DNA cycle from the cell growth cycle. Continued increase in cell mass while DNA replication is delayed would result in the abnormal size of megaloblasts. The accumulation of abnormally large cells consequent on DNA synthesis inhibition has been demonstrated in mouse lymphoma cells [35] and in the fission yeast Schizosachcharomyces pombe [36]. Although the data of Fig. 3d show that the sedimentation patterns of pulselabelled DNA chased for 24 h are very similar in untreated and vitamin-treated lymphocytes from a patient with megaloblastic anaemia, it is still possible that small regions of the chromosomes remain unreplicated in the untreated cells. If incompletely replicated chromosomes are subjected to mitotic segregation they are likely to suffer breakage at anaphase.

Acknowledgment We would like to thank the Medical Research Council for financial support.

58 References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

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