Volume 5 Number 11 Novmber 1978
Nucleic Acids Research
Incorporation of labeled degrdation products of radioactive thymine into non DNA material
Margret L.M.Anderson
nImperial Cancer Research Fund, Mil Hill Laboratoies, Burtonhole Lane, London, NW7 IAD UK Received 15- August 1978 ABSTRACT When thymine auxotrophs are grown in the presence of methyl labelled [3s] or [14C] thymine which has been stored at 4°C, two classes of material are labelled which are not DNA. One class sediments on neutral sucrose gradients with spontaneously, single stranded Okazaki pieces, is unstable in alkali, migrates on alkaline gels as very small material and is digested by ribonucleases and micrococcal nuclease, but not by DNAase I. This class is presumably RNA. The second class sediments more slowly on both neutral and alkaline sucrose gradients than Okazaki pieces, but co-migrates on alkaline gels with DNA whose size is between 700 and 4000 nucleotides. It is not digested by alkali, ribonucleases or deoxyribonucleases. Its identity is unknown. The proportion of the total incorporated counts in these two classes depends on the time of storage of the thymine and is already sufficient to interfere with certain types of experiments when the thymine is only a few weeks old. Thymine is easily purified by paper chromatography and this purified thymine does not label the two non DNA classes of material. It is recommended that radioactive thymine be purified in this way before use.
NTRODUCTICN When radioactive thymine is fed to cells, uptake of label into TCA insoluble material is usually taken as evidence of DNA synthesis. During the course of studies of the size distribution of E.coli DNA, it was observed that certain batches of radioactive thymine caused cells to accumulate excessive quantities of what appeared to be small DNA molecules. The phenomenon was investigated further. It was found that radiation induced decay products, which accumulate on storage of [3H1 and [14C] thymine label two classes of high molecular weight molecules which are not DNA. One class appears to be RNA, but the identity of the other is -unknown. The behaviour of these molecules on sucrose gradient centrifugation and on alkaline gel electrophoresis could lead to their mistaken identification as short chain DNA if no other criteria were used. The significance of these results to studies on DNA replication is discuseBd.
C Informaton Rerieval Limited I Falconbeg Court London W1 V 6FG England
4343
Nucleic Acids Research MATERIALS
[Methyl-3H] thymine (21 Ci/mmol), [methyl-14C] thymine (19 mCi/mrml) _and [methyl-3H] thymidine (24 Ci/mmol) were purchased :from the Radiochemic&l [Methyl-3H] thymine (16.7 Ci/mmol
Centre, Amersham.
had been stored for
over two years at
induced degradation products,
T2
was
900C
from Sigma.
was
RNAase I (1
were very
rich in radiation
products of the Radiochemical Centre and
were
obtained from M. Masters.
were
40C and
and 22.8 Ci/mmol) which
All the isotopes
were
mg/ml) which had
been treated for 10
stored at 4 C.
RNAase
min
at
the gift of J. Williams.
METHODS
Strains and Culture Methods E.coli W3110 (thy-)
was
from the stock of J. Cairns.
30°C with in aeration in the TES, salts medium conditions the generation time
as
Cells
described.
at
were grown
Under these
about 65 min.
was
Purification of Radioactive Thymine
Radioactive thymine
was
purified by descending chromatography
on
Whatman
3MM chromatography paper in either n-butanol/water (86/14 by vol) for 12-16 h or, more
often, in ethyl acetate saturated with O.1M sodium phosphate (pH 6.0)
for about 4 h.
Thymine migrated with
an
Rf of about 0.58 in the n-butanol
solvent and 0.22 in the ethyl acetate solvent.
[3H]
For
thymine, elution
carried out with complete medium, i.e., containing 4 -pg thymine/ml.
[14C] thymine, the elution
was
was
For
with medium lacking thymine.
Uniform Labelling of Cells
(1) Stored Radioactive Thymine. to 1
x
107
labelling
Overnight cultures of cells were diluted
cells/ml in fresh medium containing 4 pg thymine/ml for or
lacking thymine for
[14C] thymine
was
14C
labelling.
Stored
[3H]
thymine or
added to give final specific activities of 30 pCi/4 'Pg/ml
and 2 pCi/4 pg/ml respectively.
It was assumed for specific activity
calculations of stored thymine that all the radioactivity
thymine.
[3H
(2) Purified Radioactive Thymine.
was in the form of
The dilution of overnight
cultures of cells and addition of newly purified radioactive thymine was as described elsewhere.
(3) Purified
[3H]
Thymine and
stored
[14C]
Thymine.
The dilution of overnight cultures and addition of purified [14C] thymine to a
specific activity of 2 pCi/4 pg/ml
then
added to
a
was as in (2).
Stored
[3H]
thymine was
specific activity of 30 pCi/4 pg/ml.
Other Methods The procedures for killing cells in an ethanol:-phenol containing solution
4344
Nucleic Acids Research harvesting and washing cells, preparing lysates and running alkaline agarose gels have been described.1 Sucrose Gradient Centrifugation Killed cells (3 x 108) were washed in 100 mM Tris-HCl (pH 7.9), 10 mM EDTA, LM NaCl, 10 mM KCN, 5% sucrose, 4 pg thymine/ml. Washed pellets were resuspended in 200 pl of the same buffer and digested for 1 h at 60C with 50 pg lysozyme. 50 pg predigested Pronase and 10 p4 10% sarkosyl were added and incubation was carried out for 2 h at 309C. For alkali treated samples,
lysates were then made 0.2M in NaOH and incubated for 22 h at 300C. Lysates were layered on to a 10.6 ml sucrose gradient (5-20%) in gradient buffer (10 mM Tris-HCl (pH 7.9), 10 mM EDTA, 1M NaCl for neutral sucrose gradients and 10 mM EDTA, 0.9M NaCl, 0.1N NaOH for alkaline sucrose gradients). At the foot of each tube was a cushion of 0.8.ml 60% sucrose in gradient buffer. Above the lysate was layered 0.3 ml 2% sucrose in gradient buffer. Gradients were centrifuged for 17 h at 24,000 revs/min at 4 C in the SW41 rotor of a Beckman ultracentrifuge. Fractions of 480 4l were collected from the top of the tubes by upwards displacement with 70% sucrose coloured with methylene
blue. Enzyme Digestions
Pancreatic RNAase 1. Reaction mixtures contained per 110 p4; 100 p4 dialysate, 2 pg bovine serum albumin and 2 pg RNAase 1 in 20 mM Tris-HCl (pH 7.9), 1 mM EDTA. Some digestions contained [3H] X-DNA (0.3 pg, 67,000 cts/min). Incubation was for 1 h at 370C. Digestion was halted by the addition of 10 ig bovine serum albumin and 5% TCA. After 1 h at OC, precipitates were collected by filtration, dried and counted for 10 min in a toluene based scintillant. RNAase T2. Reaction mixtures contained in 120 p4; 100 p4 dialysate, 2 jg bovine serum albumin and 0.1 unit RNAase T2 in 67 mM sodium acetate (pH 4.5), 2.5 mM EDTA. Incubation was for 1 h at 37 0C. TCA insoluble counts were measured as above.
RESULTS
Labelling of Material by Stored, but not by Purified Radioactive Thymine A culture of E.coli W3110 was grown for 5 generations in medium containing stored [3H] thymine, and the cells were killed, harvested and washed. Lysates were prepared, treated with alkali and run on alkaline agarose gels. Figure l(a) shows the lbwer molecular weight end of the radioactivity distribution obtained.
It can be seen that there are two
peaks of counts 4345
Nucleic Acids Research
0*6 *
0
0*4
@0
%
0*2
0
Ib)
0*6 to
0*4 0*2
{c)0 0*6
0.4 0
7C
60
*. 50
0-2
0
40 30 Slice Number
20
10
TOD
FIGURE 1. Gel electrophoresis of lysates from uniformly labelled and chased cells of E.coli W3110. The distribution of counts following uniform labelling of cells for 5 generations with 2 year old stored [3H] thymine which had not (a) or had (b) been purified chromatographically. (c) Cells uniformly labelled as in (a) were chased in unlabelled conditioned medium for 20 min prior to preparation of lysates. Total cts/min on gels; (a) 662,600: (b) 675,800: (c) 775,200. Gels were run for 20.25 h at 20v. The short horizontal bar shows the position of the tracking dye. The long horizontal bar shows the region of the gel containing DNA chains in the range of 700-9000 nucleotides. (a &
0) migrating ahead
of the bulk of the DNA.
When a
portion of the same
[3H] thymine was purified chromatographically prior to labelling the cells, the two peaks of fast migrating material were no longer present (Figure lb). batch of
To determine if the material in peaks a & B was chaseable, cells whicn had been growing for 5 generations in medium containing stored [3H] thymine were collected on a Millipore filter, washed free of label and resuspended and grown for a further 20 min in unlabelled conditioned medium. The gel profile of a lysate of the cells is shown in Figure l(c). It is clear that 4346
Nucleic Acids Research the fast migrating material did not chase. The same result was obtained when the time of chasing was prolonged to 90 min i.e., l1 generations growth. Hence this material is stable in the cell. Characteristic of Labelling The mobility of the two peaks of fast migrating material did not depend on the particular batch of thymine used to label the cells. The slower moving peak (8) migrated to roughly the same position as DNA with chain lengths between 700 and 4000 nucleotides.
This is about the same size range
as Okazaki pieces, the DNA molecules thought to be intermediates in DNA
replication.
The faster moving material (a), migrated at roughly the same
rate as the nucleotide dTDP. The proportion of counts on a gel in the peaks a and 8 depended on the amount of impurities in the radioactive thymine used to label the cells.
This in turn depended on how long the thymine had been stored.
Thymine newly delivered from the manufacturer showed little if any radiochemical impurities on chromatographic analysis and when fed to cells, did not label any material which ran fast on gels. Within only a few weeks of first opening an ampoule of [3H] thymine, labelled impurities could be detected in the solution and
cells growing in this thymine synthesised labelled fast migrating material, to the extent of about 0.5% of the total incorporated counts. In the most extreme case found, a solution of [3H] thymine which was over two years old contained only 29% of the radioactivity as thymine. Cells growing in this preparation accumulated 29% of the total incorporated counts in peak a and 19% in peak 8. In general it> was found that the older the thymine was the more radiochemically impure it was and the higher was the proportion of total incorporated counts subsequently found in the two fast migrating peaks on gels. The ratio of counts in peaks a and 8 was not constant, but the factors affecting the ratio are unknown. The same results were obtained not only with stored [3H] thymine, but also with stored [14C] thymine.. All the results described in this paper were obtained with E.coli W3110, a wild type strain, but the same results were also found with every cell type tested including; 15T , wild type: KS463, wild type: p3478, (poLAl); RS5065, (poLAlex2); N2216, (ligts7). Thus the phenomenon is not confined to any particular strain. The kinetics of labelling of the fast migrating material were investigated. Results showed that the material was efficiently labelled by pulses of a few minutes and that the incorporation increased with time up to at least 6 generations of growth. Continuous incorporation shows that the
4347
Nucleic Acids Research radioactive compound which labels fast migrating material is not rapidly cleared from the medium and also suggests that cells do not become resistant to uptake of the compound.
Determination if Labelled Material is DNA An experiment was carried out to determine if the material labelled only by stored radioactive thymine was DNA. The design of the experiment was based on the following rationale. Cells grown in purified [14C] thymine have no labelled material which migrates fast on gels whereas cells grown in stored [3H] thymine do.
If cells are grown in a mixture of purified
[14C]
thymine and stored [3H] thymine, 14C migrating material only if that material is DNA. Cells were labelled by 5 generations growth in medium supplemented by stored [3H] thymine (Figure 2a) or [14C] thymine which had been
counts will appear in peaks of fast
1.5
1*0 0.5
SP
-
1.5
1.0 No
*
.00
0 *
0.5
. 004 00
1.5
* 0:
1-0
~~~0v
@0.
0.5
0o
70
60
50 Slice
30 40 Number
20
10
FIGURE 2. Determination if fast migrating material is DNA. The distribution thymine; of counts following uniform labelling of cells with (a) stored [3P (b) purified [14C] thymine; (c) a mixture of stored 13H] thymine and purified Total cts/min on gels: (a) [14c] thymine. (o-o-o) 14C ; (.-e-.) 3 97,000; (b)240,000; (c)220,000 [14C], 87,000 [3H].Gels were run for 22 hat 20v.
4348
Nucleic Acids Research chromatographically purified (Figure 2b) or a mixture of stored [3H] thymine and purified [14C] thymine (Figure 2c). Cells were killed and lysates were prepared and run on gels as usual. It is clear that when cells are grown in a mixture of purified [14C] thymine and stored [3H] thymine, 14C counts do not appear in either of the two peaks of fast migrating material. This shows that the fast migrating materials do not contain any thymine residues and so are not DNA. The Radioactive Compound which Labels Fast Migrating Material Given that the fast migrating material does not contain any thymine residues yet is radioactivity labelled, then it follows that the source of label cannot be thymine itself, but is presumably one or more of the radioactive impurities which accumulate in solutions of labelled thymine. In fact the impurity is not incorporated by the same pathway as thymine because the level of incorporation is not altered by raising the concentration of unlabelled thymine in the growth medium from 2 pg/ml to 50 pg/ml. Attempts were made to determine the nature of the incorporated impurity by isolating it chromatographically from solutions of stored radioactive thymine. Unfortunately it proved to be unstable. By merely spotting the stored thymine preparation on paper, drying it and immediately eluting it and feeding it to cells, the incorporation of counts into fast migrating material was reduced by about 98%. After chromatography, incorporation was completely abolished. This means that if the incorporated impurity is to be identified it will have to be by some method other than by paper chromatography. Sucrose Gradient Centrifugation Studies With a view to studying its properties, attempts were made to isolate fast migrating material from gels. This resulted in consistently low yields of material in peak 8, so experiments were carried out using sucrose gradient
centrifugation (Figure 3). When labelling was with stored radioactive thymine, neutral sucrose gradient centrifugation showed two slowly sedimenting peaks of counts, 81 (fractions 1-2), and al (fractions 4-13) which were not present if the thymine had first been purified. (Compare the 3H and 14C profiles of Figure 3(a)). To allow a comparison with short chain DNA, Figure 3(c) shows the profile of DNA labelled with a 10 sec pulse of [3H] thymidine. The spontaneously single stranded Okazaki pieces fall in fractions 4-13. It is clear that the region of the gradient encompassing the Okazaki pieces would include the material labelled by impure radioactive thymine. On alkaline sucrose gradient centrifugation, lysates of cells labelled 4349
Nucleic Acids Research
6
6
1.
4
2
2 VI-
0
0
cI
U
12
12
8
8
4
4
25
20
15
10
5
25
20
15
10
5
Slice Number FIGURE 3. Sucrose gradient centrifugation of lysates from uniformly and pulse labelled cells. 3 x 108 cells uniformly labelled with stored [3H] thymine were killed and mixed with 2 x 108 cells uniformly labelled with purified (14C] thymine. A lysate of half the cells was run on a neutral sucrose gradient (a), and a lysate of the other half was run on an alkaline sucrose gradient.(b)An unlabelled culture of 5 x 108 cells was exposed to 13H] thymidine for 10 sec then killed. A lysate of half the cells was run on a neutral sucrose gradient (c), and a lysate of the other half was run on an alkaline sucrose gradient (d). 26 fractions were collected from each gradient, but only the top 24 are shown. (-_--) 14C; (o-o-o) 3H. Total cts/min on gradients; (a) 50,000 14C, 85100 3H; (b) 71,000 14C; 85,800
3H;
(c) 8,000; (d) 8,100.
with stored radioactive thymine showed only one slowly sedimenting TCA insoluble peak, y, (fractions 1-2) and this was absent if labelling was with
purified thymine (Figure 3(b)). The material in this peak sedimented more slowly on alkaline gradients than Okazaki pieces labelled by a 10 sec pulse of [3H] thymidine (Figure 3(d)), and hence would probably not be mistaken for them.
Flectrophoresis of Gradient Peaks The mobility of the material in the slow sedimenting peaks of gradients was examined on gels of the type shown in Figure 1. Peak al of the neutral 4350
Nucleic Acids Research sucrose gradient co-migrated with the faster migrating peak a, while peak 81 co-migrated with the slower migrating peak 8 (data not shown). With peak y from the alkaline sucrose gradient, about 33% of the counts migrated with
peak a and about 60% with peak 8. Alkali Lability of Peak al Peak al from the neutral sucrose gradient behaves as high molecular weight material on neutral gradients and as low molecular weight material on alkaline gels. This re.sult suggests that the material is alkali labile.
This was confirmed by treating an aliquot of peak al from a neutral sucrose gradient with 0.3M NaOH for 18 h at 370C, neutralising with HC1 and measuring TCA insoluble counts. 451 cts/min above background were present in the un-
treated control whereas only 29 were present after treatment with alkali. This result explains why very fast migrating material is found when cell lysates are run on alkaline gels. Clearly the molecules as they exist in the cells are much larger, but are broken down on exposure to alkali and consequently migrate fast on gels. The finding of both classes of material at the top of alkaline sucrose gradients is also consistent with the alkali lability of the higher molecular weight species. Determination if Material in Gradient Peaks is DNA Material from the slowly sedimenting peaks on sucrose gradients was tested for its susceptibility to digestion by various nucleases. Dialysates of peaks 81 and y were resistant to digestion by both pancreatic DNAase I and micrococcal nuclease under condition where [14C] A-DNA was digested to over 90% of the original counts. It is unlikely that a nuclease inhibitor in the dialysates prevented the enzymes acting since the [14C] A-DNA was digested to the same extent in the presence of each dialysate. Material recovered from peak al was not digested by DNAase I, but on treatment with micrococcal nuclease the TCA insoluble counts fell from 682 cts/min to 58 cts/min. Since this enzyme digests RNA as well as DNA, it seemed possible that the material in peak al might be RNA. RNAase digestion studies were carried out (Table 1) and it was found that the material from peak al was digested by both pancreatic RNAase I and RNAase T2. This result together with that of the alkali lability studies indicates either that the material in peak al is RNA itself or else that the radioactive component is attached to RNA (e.g., similar to the way that amino acid is attached to RNA in amino acyl-tRNA). The material from the top of both neutral and alkaline sucrose gradients was resistant to RNAase digestion. The identity of this slower sedimenting
4351
Nucleic Acids Research TABLE 1
Treatment of gradient peaks with RNAases TCA insoluble cts/min
pancreatic RNAase I
RNAase T2
.;aterials for digestion Neutral gradient
peak 81 peak al lkaline gradient peak y
[3H] A-DNA
276
259
289
49
163 342
151 70
120 6718
130
-
-
7081
-
-
material (peak 81) is unknown, but it would appear not to be a nucleic acid. CONCLUSIONS The stimulus for undertaking the present study was the finding that on repeated analysis of the size distribution of short chain DNA in E.coli, there was considerable variability in the results. The cause was traced to the radioactivity thymine used to label the DNA. As the experiments here show, unless the thymine is radiochemically pure, two non chaseable classes of material are labelled in addition to the DNA. The mistaken identification of the slower migrating of these classes as DNA can account for the
variability noted above. One class of molecule labelled by impure radioactive thymine is apparently RNA. This identification is based on the observations that the material is broken down on exposure to alkali, is resistant to digestion by DNAase 1, but is digested by micrococcal nuclease and by ribonucleases. As the results here show, the RNA sediments on neutral sucrose gradients at the same rate as spontaneously single stranded Okazaki pieces. Since it is common practice to heat treat pulse labelled DNA and run it on neutral sucrose gradients in order to isolate DNA replication intermediates, it is obviously undersirable to have contamination by other species of labelled molecules. On treatment with alkali, these RNA molecules break down to give TCA soluble products so that they are not usually detected on a alkaline sucrose gradients. However it is important to note that the breakdown products are still present at the top of gradients and are not completely 4352
Nucleic Acids Research removed by dialysis.
On alkaline gels where total counts
analysed, the
are
breakdown products migrate well ahead of short chain DNA and
so
should not be
mistaken for DNA. The labelling of RNA by thymine with ring might be predicted since thymine a precursor
of RNA.
a
can
radioactive atom in the pyrimidine be demethylated to uracil which is
But the labelling of RNA by
a
derivative of methyl
labelled thymine would probably not have been expected, although it has been reported that breakdown products of methyl labelled thymidine
can
be incor-
porated into proteins.2 The
identity of the second class of molecule which is labelled by impure
radioactive thymine is unknown. since lysates
likely to be
are a
It does not
appear to
be
a
nucleic acid and
treated with Pronase before application to gels, it is
protein.
un-
Its electrophoretic mobility is sufficiently like
that of short chain DNA for it to be mistaken the other hand, its sedimentation
on
as
such
on
alkaline gels.
both neutral and alkaline
gradients is much slower than Okazaki pieces
so
On
sucrose
it is unlikely that it would
be mistaken for them using these techniques. Chromatography of thymine preparations which labelled the two
non
DNA
classes of material revealed at least five other radioactive species besides
thymine.
Although
a
direct proof is lacking because of their instability to
chromatography, it is presumably
one or more
The fact that
the non DNA material.
no
of these species which labels
such material is labelled when the
thymine is purified supports this conclusion. It is well documented that radioactive compounds undergo radiolysis
on
In many types of experiment the presence of labelled degradation does not seem to interfere and it may not be necessary to purify radioactive
storage. '
compounds before
In others however, it is essential to use purified
use.
labelled compounds.
For example, autoradiographic studies have shown that
breakdown products of
[3H]
Tetrahymena pyriformis and acid in the cytoplasm.
thymidine are
are
rapidly taken up by cells of
incorporated into molecules other than nucleic
Further, there is evidence that minor contaminants
in preparations of [3H] thymidine labelled in the methyl position enter DNA
of Chlamydomonas rheinhardtii with kinetics which differ from that of In addition as results here show, in experiments where thymidine itself. short chain DNA is analysed by gel electrophoresis or sucrose gradient centrifugation, it is essential to use radiochemically pure thymine for
labelling. It might be expected that thymine preparations stored for as long as two
4353
Nucleic Acids Research years would be grossly contaminated with radiolysis products and that these compounds might be incorporated into macromolecules. It would probably not have been predicted that labelled compounds which had been opened for only about a month (normally considered an acceptable time of storage) would have given rise to problems. In this case the proportion of total counts incorporated into material other than DNA is about 0.5%. It is important to emphasise that although this value is small, it is not negligible. Short If only chain DNA accounts for only about 1% of the total DNA of the cell. that region of gels or gradients containing short chain DNA is considered,. then the contribution by non DNA molecules is equal to or greater than the
contribution by DNA itself. The presence of small amounts of radioactive impurities in relatively fresh preparations of labelled thymine could also cause misinterpretation of results in experiments where the normal level of DNA synthesis has been reduced e.g., in studies of residual DNA synthesis after temperature sensitive mutants have been placed at the restrictive temperature or after DNA replication inhibitors have been administered. Paper chromatography is a simple and convenient method for purifyingj labelled thymine and is effective even on old preparations which have accumulated more than half the radioactivity in degradation products. It is recommended that all radioactive thymine preparations regardless of age be
purified in this way before use. ACKNOWLEDGEMMTS I should like to thank S. Apelgot, J. Cairns and I.M. Kerr for discussions and J. Williams for gifts of enzymes. * Present address: Beatson Institute, Garscube Estate,
Bearsden Road,
Glasgow. REFERENCES 1. Anderson, M.L.M. (1978) J. Iol. Biol. 118, 227-240. 2. Bryant, B.J. (1966) J. Cell Biol. 29, 29-36. 3. Bayly, R.J. and Evans, E.A. (1969) in Handbook of Radioactive Nuclides, pp. 285, The Chemical Rubber Co., Ohio. 4. Evans, E.A. (1966) in Tritium and its Compounds, Butterworth, London. 5. Wand, M., Zeuthen, E. and Evans, E.A. (1967) Science, 157, 436-438. 6. Dashe, S.W. and Howell, S.H. (1976) J. Cell Biol. 69, 215-218.
4364
Nucleic Acids Research
Incorporation of labeled degrdation products of radioactive thymine into non DNA material
Margret L.M.Anderson
nImperial Cancer Research Fund, Mil Hill Laboratoies, Burtonhole Lane, London, NW7 IAD UK Received 15- August 1978 ABSTRACT When thymine auxotrophs are grown in the presence of methyl labelled [3s] or [14C] thymine which has been stored at 4°C, two classes of material are labelled which are not DNA. One class sediments on neutral sucrose gradients with spontaneously, single stranded Okazaki pieces, is unstable in alkali, migrates on alkaline gels as very small material and is digested by ribonucleases and micrococcal nuclease, but not by DNAase I. This class is presumably RNA. The second class sediments more slowly on both neutral and alkaline sucrose gradients than Okazaki pieces, but co-migrates on alkaline gels with DNA whose size is between 700 and 4000 nucleotides. It is not digested by alkali, ribonucleases or deoxyribonucleases. Its identity is unknown. The proportion of the total incorporated counts in these two classes depends on the time of storage of the thymine and is already sufficient to interfere with certain types of experiments when the thymine is only a few weeks old. Thymine is easily purified by paper chromatography and this purified thymine does not label the two non DNA classes of material. It is recommended that radioactive thymine be purified in this way before use.
NTRODUCTICN When radioactive thymine is fed to cells, uptake of label into TCA insoluble material is usually taken as evidence of DNA synthesis. During the course of studies of the size distribution of E.coli DNA, it was observed that certain batches of radioactive thymine caused cells to accumulate excessive quantities of what appeared to be small DNA molecules. The phenomenon was investigated further. It was found that radiation induced decay products, which accumulate on storage of [3H1 and [14C] thymine label two classes of high molecular weight molecules which are not DNA. One class appears to be RNA, but the identity of the other is -unknown. The behaviour of these molecules on sucrose gradient centrifugation and on alkaline gel electrophoresis could lead to their mistaken identification as short chain DNA if no other criteria were used. The significance of these results to studies on DNA replication is discuseBd.
C Informaton Rerieval Limited I Falconbeg Court London W1 V 6FG England
4343
Nucleic Acids Research MATERIALS
[Methyl-3H] thymine (21 Ci/mmol), [methyl-14C] thymine (19 mCi/mrml) _and [methyl-3H] thymidine (24 Ci/mmol) were purchased :from the Radiochemic&l [Methyl-3H] thymine (16.7 Ci/mmol
Centre, Amersham.
had been stored for
over two years at
induced degradation products,
T2
was
900C
from Sigma.
was
RNAase I (1
were very
rich in radiation
products of the Radiochemical Centre and
were
obtained from M. Masters.
were
40C and
and 22.8 Ci/mmol) which
All the isotopes
were
mg/ml) which had
been treated for 10
stored at 4 C.
RNAase
min
at
the gift of J. Williams.
METHODS
Strains and Culture Methods E.coli W3110 (thy-)
was
from the stock of J. Cairns.
30°C with in aeration in the TES, salts medium conditions the generation time
as
Cells
described.
at
were grown
Under these
about 65 min.
was
Purification of Radioactive Thymine
Radioactive thymine
was
purified by descending chromatography
on
Whatman
3MM chromatography paper in either n-butanol/water (86/14 by vol) for 12-16 h or, more
often, in ethyl acetate saturated with O.1M sodium phosphate (pH 6.0)
for about 4 h.
Thymine migrated with
an
Rf of about 0.58 in the n-butanol
solvent and 0.22 in the ethyl acetate solvent.
[3H]
For
thymine, elution
carried out with complete medium, i.e., containing 4 -pg thymine/ml.
[14C] thymine, the elution
was
was
For
with medium lacking thymine.
Uniform Labelling of Cells
(1) Stored Radioactive Thymine. to 1
x
107
labelling
Overnight cultures of cells were diluted
cells/ml in fresh medium containing 4 pg thymine/ml for or
lacking thymine for
[14C] thymine
was
14C
labelling.
Stored
[3H]
thymine or
added to give final specific activities of 30 pCi/4 'Pg/ml
and 2 pCi/4 pg/ml respectively.
It was assumed for specific activity
calculations of stored thymine that all the radioactivity
thymine.
[3H
(2) Purified Radioactive Thymine.
was in the form of
The dilution of overnight
cultures of cells and addition of newly purified radioactive thymine was as described elsewhere.
(3) Purified
[3H]
Thymine and
stored
[14C]
Thymine.
The dilution of overnight cultures and addition of purified [14C] thymine to a
specific activity of 2 pCi/4 pg/ml
then
added to
a
was as in (2).
Stored
[3H]
thymine was
specific activity of 30 pCi/4 pg/ml.
Other Methods The procedures for killing cells in an ethanol:-phenol containing solution
4344
Nucleic Acids Research harvesting and washing cells, preparing lysates and running alkaline agarose gels have been described.1 Sucrose Gradient Centrifugation Killed cells (3 x 108) were washed in 100 mM Tris-HCl (pH 7.9), 10 mM EDTA, LM NaCl, 10 mM KCN, 5% sucrose, 4 pg thymine/ml. Washed pellets were resuspended in 200 pl of the same buffer and digested for 1 h at 60C with 50 pg lysozyme. 50 pg predigested Pronase and 10 p4 10% sarkosyl were added and incubation was carried out for 2 h at 309C. For alkali treated samples,
lysates were then made 0.2M in NaOH and incubated for 22 h at 300C. Lysates were layered on to a 10.6 ml sucrose gradient (5-20%) in gradient buffer (10 mM Tris-HCl (pH 7.9), 10 mM EDTA, 1M NaCl for neutral sucrose gradients and 10 mM EDTA, 0.9M NaCl, 0.1N NaOH for alkaline sucrose gradients). At the foot of each tube was a cushion of 0.8.ml 60% sucrose in gradient buffer. Above the lysate was layered 0.3 ml 2% sucrose in gradient buffer. Gradients were centrifuged for 17 h at 24,000 revs/min at 4 C in the SW41 rotor of a Beckman ultracentrifuge. Fractions of 480 4l were collected from the top of the tubes by upwards displacement with 70% sucrose coloured with methylene
blue. Enzyme Digestions
Pancreatic RNAase 1. Reaction mixtures contained per 110 p4; 100 p4 dialysate, 2 pg bovine serum albumin and 2 pg RNAase 1 in 20 mM Tris-HCl (pH 7.9), 1 mM EDTA. Some digestions contained [3H] X-DNA (0.3 pg, 67,000 cts/min). Incubation was for 1 h at 370C. Digestion was halted by the addition of 10 ig bovine serum albumin and 5% TCA. After 1 h at OC, precipitates were collected by filtration, dried and counted for 10 min in a toluene based scintillant. RNAase T2. Reaction mixtures contained in 120 p4; 100 p4 dialysate, 2 jg bovine serum albumin and 0.1 unit RNAase T2 in 67 mM sodium acetate (pH 4.5), 2.5 mM EDTA. Incubation was for 1 h at 37 0C. TCA insoluble counts were measured as above.
RESULTS
Labelling of Material by Stored, but not by Purified Radioactive Thymine A culture of E.coli W3110 was grown for 5 generations in medium containing stored [3H] thymine, and the cells were killed, harvested and washed. Lysates were prepared, treated with alkali and run on alkaline agarose gels. Figure l(a) shows the lbwer molecular weight end of the radioactivity distribution obtained.
It can be seen that there are two
peaks of counts 4345
Nucleic Acids Research
0*6 *
0
0*4
@0
%
0*2
0
Ib)
0*6 to
0*4 0*2
{c)0 0*6
0.4 0
7C
60
*. 50
0-2
0
40 30 Slice Number
20
10
TOD
FIGURE 1. Gel electrophoresis of lysates from uniformly labelled and chased cells of E.coli W3110. The distribution of counts following uniform labelling of cells for 5 generations with 2 year old stored [3H] thymine which had not (a) or had (b) been purified chromatographically. (c) Cells uniformly labelled as in (a) were chased in unlabelled conditioned medium for 20 min prior to preparation of lysates. Total cts/min on gels; (a) 662,600: (b) 675,800: (c) 775,200. Gels were run for 20.25 h at 20v. The short horizontal bar shows the position of the tracking dye. The long horizontal bar shows the region of the gel containing DNA chains in the range of 700-9000 nucleotides. (a &
0) migrating ahead
of the bulk of the DNA.
When a
portion of the same
[3H] thymine was purified chromatographically prior to labelling the cells, the two peaks of fast migrating material were no longer present (Figure lb). batch of
To determine if the material in peaks a & B was chaseable, cells whicn had been growing for 5 generations in medium containing stored [3H] thymine were collected on a Millipore filter, washed free of label and resuspended and grown for a further 20 min in unlabelled conditioned medium. The gel profile of a lysate of the cells is shown in Figure l(c). It is clear that 4346
Nucleic Acids Research the fast migrating material did not chase. The same result was obtained when the time of chasing was prolonged to 90 min i.e., l1 generations growth. Hence this material is stable in the cell. Characteristic of Labelling The mobility of the two peaks of fast migrating material did not depend on the particular batch of thymine used to label the cells. The slower moving peak (8) migrated to roughly the same position as DNA with chain lengths between 700 and 4000 nucleotides.
This is about the same size range
as Okazaki pieces, the DNA molecules thought to be intermediates in DNA
replication.
The faster moving material (a), migrated at roughly the same
rate as the nucleotide dTDP. The proportion of counts on a gel in the peaks a and 8 depended on the amount of impurities in the radioactive thymine used to label the cells.
This in turn depended on how long the thymine had been stored.
Thymine newly delivered from the manufacturer showed little if any radiochemical impurities on chromatographic analysis and when fed to cells, did not label any material which ran fast on gels. Within only a few weeks of first opening an ampoule of [3H] thymine, labelled impurities could be detected in the solution and
cells growing in this thymine synthesised labelled fast migrating material, to the extent of about 0.5% of the total incorporated counts. In the most extreme case found, a solution of [3H] thymine which was over two years old contained only 29% of the radioactivity as thymine. Cells growing in this preparation accumulated 29% of the total incorporated counts in peak a and 19% in peak 8. In general it> was found that the older the thymine was the more radiochemically impure it was and the higher was the proportion of total incorporated counts subsequently found in the two fast migrating peaks on gels. The ratio of counts in peaks a and 8 was not constant, but the factors affecting the ratio are unknown. The same results were obtained not only with stored [3H] thymine, but also with stored [14C] thymine.. All the results described in this paper were obtained with E.coli W3110, a wild type strain, but the same results were also found with every cell type tested including; 15T , wild type: KS463, wild type: p3478, (poLAl); RS5065, (poLAlex2); N2216, (ligts7). Thus the phenomenon is not confined to any particular strain. The kinetics of labelling of the fast migrating material were investigated. Results showed that the material was efficiently labelled by pulses of a few minutes and that the incorporation increased with time up to at least 6 generations of growth. Continuous incorporation shows that the
4347
Nucleic Acids Research radioactive compound which labels fast migrating material is not rapidly cleared from the medium and also suggests that cells do not become resistant to uptake of the compound.
Determination if Labelled Material is DNA An experiment was carried out to determine if the material labelled only by stored radioactive thymine was DNA. The design of the experiment was based on the following rationale. Cells grown in purified [14C] thymine have no labelled material which migrates fast on gels whereas cells grown in stored [3H] thymine do.
If cells are grown in a mixture of purified
[14C]
thymine and stored [3H] thymine, 14C migrating material only if that material is DNA. Cells were labelled by 5 generations growth in medium supplemented by stored [3H] thymine (Figure 2a) or [14C] thymine which had been
counts will appear in peaks of fast
1.5
1*0 0.5
SP
-
1.5
1.0 No
*
.00
0 *
0.5
. 004 00
1.5
* 0:
1-0
~~~0v
@0.
0.5
0o
70
60
50 Slice
30 40 Number
20
10
FIGURE 2. Determination if fast migrating material is DNA. The distribution thymine; of counts following uniform labelling of cells with (a) stored [3P (b) purified [14C] thymine; (c) a mixture of stored 13H] thymine and purified Total cts/min on gels: (a) [14c] thymine. (o-o-o) 14C ; (.-e-.) 3 97,000; (b)240,000; (c)220,000 [14C], 87,000 [3H].Gels were run for 22 hat 20v.
4348
Nucleic Acids Research chromatographically purified (Figure 2b) or a mixture of stored [3H] thymine and purified [14C] thymine (Figure 2c). Cells were killed and lysates were prepared and run on gels as usual. It is clear that when cells are grown in a mixture of purified [14C] thymine and stored [3H] thymine, 14C counts do not appear in either of the two peaks of fast migrating material. This shows that the fast migrating materials do not contain any thymine residues and so are not DNA. The Radioactive Compound which Labels Fast Migrating Material Given that the fast migrating material does not contain any thymine residues yet is radioactivity labelled, then it follows that the source of label cannot be thymine itself, but is presumably one or more of the radioactive impurities which accumulate in solutions of labelled thymine. In fact the impurity is not incorporated by the same pathway as thymine because the level of incorporation is not altered by raising the concentration of unlabelled thymine in the growth medium from 2 pg/ml to 50 pg/ml. Attempts were made to determine the nature of the incorporated impurity by isolating it chromatographically from solutions of stored radioactive thymine. Unfortunately it proved to be unstable. By merely spotting the stored thymine preparation on paper, drying it and immediately eluting it and feeding it to cells, the incorporation of counts into fast migrating material was reduced by about 98%. After chromatography, incorporation was completely abolished. This means that if the incorporated impurity is to be identified it will have to be by some method other than by paper chromatography. Sucrose Gradient Centrifugation Studies With a view to studying its properties, attempts were made to isolate fast migrating material from gels. This resulted in consistently low yields of material in peak 8, so experiments were carried out using sucrose gradient
centrifugation (Figure 3). When labelling was with stored radioactive thymine, neutral sucrose gradient centrifugation showed two slowly sedimenting peaks of counts, 81 (fractions 1-2), and al (fractions 4-13) which were not present if the thymine had first been purified. (Compare the 3H and 14C profiles of Figure 3(a)). To allow a comparison with short chain DNA, Figure 3(c) shows the profile of DNA labelled with a 10 sec pulse of [3H] thymidine. The spontaneously single stranded Okazaki pieces fall in fractions 4-13. It is clear that the region of the gradient encompassing the Okazaki pieces would include the material labelled by impure radioactive thymine. On alkaline sucrose gradient centrifugation, lysates of cells labelled 4349
Nucleic Acids Research
6
6
1.
4
2
2 VI-
0
0
cI
U
12
12
8
8
4
4
25
20
15
10
5
25
20
15
10
5
Slice Number FIGURE 3. Sucrose gradient centrifugation of lysates from uniformly and pulse labelled cells. 3 x 108 cells uniformly labelled with stored [3H] thymine were killed and mixed with 2 x 108 cells uniformly labelled with purified (14C] thymine. A lysate of half the cells was run on a neutral sucrose gradient (a), and a lysate of the other half was run on an alkaline sucrose gradient.(b)An unlabelled culture of 5 x 108 cells was exposed to 13H] thymidine for 10 sec then killed. A lysate of half the cells was run on a neutral sucrose gradient (c), and a lysate of the other half was run on an alkaline sucrose gradient (d). 26 fractions were collected from each gradient, but only the top 24 are shown. (-_--) 14C; (o-o-o) 3H. Total cts/min on gradients; (a) 50,000 14C, 85100 3H; (b) 71,000 14C; 85,800
3H;
(c) 8,000; (d) 8,100.
with stored radioactive thymine showed only one slowly sedimenting TCA insoluble peak, y, (fractions 1-2) and this was absent if labelling was with
purified thymine (Figure 3(b)). The material in this peak sedimented more slowly on alkaline gradients than Okazaki pieces labelled by a 10 sec pulse of [3H] thymidine (Figure 3(d)), and hence would probably not be mistaken for them.
Flectrophoresis of Gradient Peaks The mobility of the material in the slow sedimenting peaks of gradients was examined on gels of the type shown in Figure 1. Peak al of the neutral 4350
Nucleic Acids Research sucrose gradient co-migrated with the faster migrating peak a, while peak 81 co-migrated with the slower migrating peak 8 (data not shown). With peak y from the alkaline sucrose gradient, about 33% of the counts migrated with
peak a and about 60% with peak 8. Alkali Lability of Peak al Peak al from the neutral sucrose gradient behaves as high molecular weight material on neutral gradients and as low molecular weight material on alkaline gels. This re.sult suggests that the material is alkali labile.
This was confirmed by treating an aliquot of peak al from a neutral sucrose gradient with 0.3M NaOH for 18 h at 370C, neutralising with HC1 and measuring TCA insoluble counts. 451 cts/min above background were present in the un-
treated control whereas only 29 were present after treatment with alkali. This result explains why very fast migrating material is found when cell lysates are run on alkaline gels. Clearly the molecules as they exist in the cells are much larger, but are broken down on exposure to alkali and consequently migrate fast on gels. The finding of both classes of material at the top of alkaline sucrose gradients is also consistent with the alkali lability of the higher molecular weight species. Determination if Material in Gradient Peaks is DNA Material from the slowly sedimenting peaks on sucrose gradients was tested for its susceptibility to digestion by various nucleases. Dialysates of peaks 81 and y were resistant to digestion by both pancreatic DNAase I and micrococcal nuclease under condition where [14C] A-DNA was digested to over 90% of the original counts. It is unlikely that a nuclease inhibitor in the dialysates prevented the enzymes acting since the [14C] A-DNA was digested to the same extent in the presence of each dialysate. Material recovered from peak al was not digested by DNAase I, but on treatment with micrococcal nuclease the TCA insoluble counts fell from 682 cts/min to 58 cts/min. Since this enzyme digests RNA as well as DNA, it seemed possible that the material in peak al might be RNA. RNAase digestion studies were carried out (Table 1) and it was found that the material from peak al was digested by both pancreatic RNAase I and RNAase T2. This result together with that of the alkali lability studies indicates either that the material in peak al is RNA itself or else that the radioactive component is attached to RNA (e.g., similar to the way that amino acid is attached to RNA in amino acyl-tRNA). The material from the top of both neutral and alkaline sucrose gradients was resistant to RNAase digestion. The identity of this slower sedimenting
4351
Nucleic Acids Research TABLE 1
Treatment of gradient peaks with RNAases TCA insoluble cts/min
pancreatic RNAase I
RNAase T2
.;aterials for digestion Neutral gradient
peak 81 peak al lkaline gradient peak y
[3H] A-DNA
276
259
289
49
163 342
151 70
120 6718
130
-
-
7081
-
-
material (peak 81) is unknown, but it would appear not to be a nucleic acid. CONCLUSIONS The stimulus for undertaking the present study was the finding that on repeated analysis of the size distribution of short chain DNA in E.coli, there was considerable variability in the results. The cause was traced to the radioactivity thymine used to label the DNA. As the experiments here show, unless the thymine is radiochemically pure, two non chaseable classes of material are labelled in addition to the DNA. The mistaken identification of the slower migrating of these classes as DNA can account for the
variability noted above. One class of molecule labelled by impure radioactive thymine is apparently RNA. This identification is based on the observations that the material is broken down on exposure to alkali, is resistant to digestion by DNAase 1, but is digested by micrococcal nuclease and by ribonucleases. As the results here show, the RNA sediments on neutral sucrose gradients at the same rate as spontaneously single stranded Okazaki pieces. Since it is common practice to heat treat pulse labelled DNA and run it on neutral sucrose gradients in order to isolate DNA replication intermediates, it is obviously undersirable to have contamination by other species of labelled molecules. On treatment with alkali, these RNA molecules break down to give TCA soluble products so that they are not usually detected on a alkaline sucrose gradients. However it is important to note that the breakdown products are still present at the top of gradients and are not completely 4352
Nucleic Acids Research removed by dialysis.
On alkaline gels where total counts
analysed, the
are
breakdown products migrate well ahead of short chain DNA and
so
should not be
mistaken for DNA. The labelling of RNA by thymine with ring might be predicted since thymine a precursor
of RNA.
a
can
radioactive atom in the pyrimidine be demethylated to uracil which is
But the labelling of RNA by
a
derivative of methyl
labelled thymine would probably not have been expected, although it has been reported that breakdown products of methyl labelled thymidine
can
be incor-
porated into proteins.2 The
identity of the second class of molecule which is labelled by impure
radioactive thymine is unknown. since lysates
likely to be
are a
It does not
appear to
be
a
nucleic acid and
treated with Pronase before application to gels, it is
protein.
un-
Its electrophoretic mobility is sufficiently like
that of short chain DNA for it to be mistaken the other hand, its sedimentation
on
as
such
on
alkaline gels.
both neutral and alkaline
gradients is much slower than Okazaki pieces
so
On
sucrose
it is unlikely that it would
be mistaken for them using these techniques. Chromatography of thymine preparations which labelled the two
non
DNA
classes of material revealed at least five other radioactive species besides
thymine.
Although
a
direct proof is lacking because of their instability to
chromatography, it is presumably
one or more
The fact that
the non DNA material.
no
of these species which labels
such material is labelled when the
thymine is purified supports this conclusion. It is well documented that radioactive compounds undergo radiolysis
on
In many types of experiment the presence of labelled degradation does not seem to interfere and it may not be necessary to purify radioactive
storage. '
compounds before
In others however, it is essential to use purified
use.
labelled compounds.
For example, autoradiographic studies have shown that
breakdown products of
[3H]
Tetrahymena pyriformis and acid in the cytoplasm.
thymidine are
are
rapidly taken up by cells of
incorporated into molecules other than nucleic
Further, there is evidence that minor contaminants
in preparations of [3H] thymidine labelled in the methyl position enter DNA
of Chlamydomonas rheinhardtii with kinetics which differ from that of In addition as results here show, in experiments where thymidine itself. short chain DNA is analysed by gel electrophoresis or sucrose gradient centrifugation, it is essential to use radiochemically pure thymine for
labelling. It might be expected that thymine preparations stored for as long as two
4353
Nucleic Acids Research years would be grossly contaminated with radiolysis products and that these compounds might be incorporated into macromolecules. It would probably not have been predicted that labelled compounds which had been opened for only about a month (normally considered an acceptable time of storage) would have given rise to problems. In this case the proportion of total counts incorporated into material other than DNA is about 0.5%. It is important to emphasise that although this value is small, it is not negligible. Short If only chain DNA accounts for only about 1% of the total DNA of the cell. that region of gels or gradients containing short chain DNA is considered,. then the contribution by non DNA molecules is equal to or greater than the
contribution by DNA itself. The presence of small amounts of radioactive impurities in relatively fresh preparations of labelled thymine could also cause misinterpretation of results in experiments where the normal level of DNA synthesis has been reduced e.g., in studies of residual DNA synthesis after temperature sensitive mutants have been placed at the restrictive temperature or after DNA replication inhibitors have been administered. Paper chromatography is a simple and convenient method for purifyingj labelled thymine and is effective even on old preparations which have accumulated more than half the radioactivity in degradation products. It is recommended that all radioactive thymine preparations regardless of age be
purified in this way before use. ACKNOWLEDGEMMTS I should like to thank S. Apelgot, J. Cairns and I.M. Kerr for discussions and J. Williams for gifts of enzymes. * Present address: Beatson Institute, Garscube Estate,
Bearsden Road,
Glasgow. REFERENCES 1. Anderson, M.L.M. (1978) J. Iol. Biol. 118, 227-240. 2. Bryant, B.J. (1966) J. Cell Biol. 29, 29-36. 3. Bayly, R.J. and Evans, E.A. (1969) in Handbook of Radioactive Nuclides, pp. 285, The Chemical Rubber Co., Ohio. 4. Evans, E.A. (1966) in Tritium and its Compounds, Butterworth, London. 5. Wand, M., Zeuthen, E. and Evans, E.A. (1967) Science, 157, 436-438. 6. Dashe, S.W. and Howell, S.H. (1976) J. Cell Biol. 69, 215-218.
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